Compositions and methods comprising histidyl-trna synthetase splice variants having non-canonical biological activities

ABSTRACT

Isolated histidyl-tRNA synthetase splice variant polynucleotides and polypeptides having non-canonical biological activities are provided, as well as compositions and methods related thereto.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.14/262,272, filed Apr. 25, 2014; which is a Continuation of U.S.application Ser. No. 13/766,659, filed Feb. 13, 2013, now U.S. Pat. No.8,753,638, issued Jun. 17, 2014; which is a Continuation of U.S.application Ser. No. 12/725,272, filed Mar. 16, 2010, now U.S. Pat. No.8,404,242, issued Mar. 26, 2013; which claims the benefit under 35U.S.C. § 119(e) of U.S. Provisional Patent Application 61/160,630, filedMar. 16, 2009, and U.S. Provisional Patent Application 61/239,747, filedSep. 3, 2009, each of which is incorporated by reference in itsentirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is ATYR_015_05US_ST25.txt. The text file is 20 KB,was created on Sep. 5, 2016, and is being submitted electronically viaEFS-Web.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates generally to histidyl-tRNA synthetase(HRS) splice variant polynucleotides and polypeptides, compositionscomprising such polynucleotides and polypeptides, and methods of usingsame.

Description of the Related Art

Aminoacyl-tRNA synthetase (AARS) proteins, which catalyze theaminoacylation of tRNA molecules, are essential for decoding geneticinformation during the process of translation. Each of the eukaryotictRNA synthetases consists of a core enzyme, which is closely related tothe prokaryotic tRNA synthetase, as well as additional domains that areappended to the amino-terminal end, carboxyl-terminal end or insertedinto a region internal to the core enzyme. Human tyrosyl-tRNA synthetase(TyrRS), for example, has a carboxyl-terminal domain that is not part ofprokaryotic and lower eukaryotic TyrRS molecules.

Several aminoacyl-tRNA synthetases have been demonstrated to havenon-canonical functions distinct from their involvement in translation.For example, Mini-tyrosyl tRNA synthetase (mini-TyrRS), the N-terminaldomain of TyrRS which corresponds to amino acid residues 1-364 and iscleaved by polymorphonuclear cell elastase and plasmin, is a member ofthe aminoacyl tRNA synthetase “AARS” multifunction cytokine-likeproteins and peptides. In vitro, Mini-TyrRS has been shown to stimulateneutrophil activation and chemotaxis, endothelial cell proliferation andmigration, and is pro-angiogenic in chick chorioallantoic membrane (CAM)and mouse matrigel assays. Mini-TyrRS has an ELR motif that, likeCXC-chemokines such as IL-8, confers its chemokine and angiogenicactivities. Like other ELR-containing cytokines, mutation of this motifinhibits mini-TyrRS binding and stimulation of leukocytes andangiogenesis.

In addition, truncated forms of TrpRS have been demonstrated to haveangiogenic properties. In normal human cells, there are two forms ofTrpRS that can be detected: a major form consisting of the full-lengthmolecule (amino acid residues 1-471) and a minor truncated form. Theminor form is generated by the deletion of an amino-terminal domainthrough alternative splicing of the pre-mRNA. The amino-terminus ofmini-TrpRS has been determined to be the methionine residue at position48 of the full-length TrpRS molecule. Alternatively, truncated TrpRS canbe generated by proteolysis. For example, bovine TrpRS is highlyexpressed in the pancreas and is secreted into the pancreatic juice,thus resulting in the production of a truncated TrpRS molecule.Additional studies indicate that mini-TrpRS inhibits VEGF-induced cellproliferation and migration (Wakasugi et al., Proc. Natl. Acad. Sci. 99:173-177 (2002)). In particular, a chick CAM assay shows that mini-TrpRSblocks angiogenic activity of VEGF. In contrast, the full-length TrpRSdoes not inhibit angiogenesis. Thus, removal of the first 48 amino acidresidues exposes the anti-angiogenic activity of TrpRS. Therefore, aswith TyrRS, certain forms of TrpRS possess activities other than theaminoacylation of tRNA.

Given these observations of non-canonical and therapeutically relevantactivities associated with alternative forms of TyrRS and TrpRS, thereis a need to identify biologically relevant forms and/or activities ofother aminoacyl-tRNA synthetase proteins in order to exploit the fulltherapeutic potential of this family of proteins. Accordingly, thepresent invention addresses these needs and offers other relatedadvantages.

SUMMARY OF THE INVENTION

The present invention relates generally to isolated HRS splice variantpolypeptides having non-canonical activities; HRS splice variantpolynucleotides encoding HRS splice variant polypeptides; binding agentsthat bind HRS polypeptides; analogs, variants and fragments of HRSpolypeptides and polynucleotides, as well as compositions and methods ofmaking and using any of the foregoing.

Therefore, according to one aspect, the present invention providesisolated HRS splice variant polypeptides having at least onenon-canonical biological activity, as well active fragments and variantsthereof. “Non-canonical” activity,” as used herein, refers generally toan activity possessed by a HRS polypeptide of the invention that isother than aminoacylation and, more specifically, other than theaddition of histidine onto a tRNA^(His) molecule. As described herein,in certain embodiments, a non-canonical biological activity exhibited bya HRS polypeptide of the invention may include, but is not limited to,modulation of cytokine production, modulation of cell proliferation,modulation of apoptosis, modulation of cell signaling, modulation ofangiogenesis, modulation of cell migration, modulation of cell binding,modulation of cellular metabolism, and the like.

In one illustrative embodiment, the HRS splice variant polypeptide ofthe invention is a HRS fragment comprising at least the WHEP domain ofHRS, e.g., amino acid residues 3-43 of the human full length HRSprotein. In another embodiment, the HRS splice variant polypeptide ofthe invention is a HRS fragment comprising at least the anticodonbinding domain of HRS, e.g., amino acid residues 406-501 of the fulllength human HRS protein. In yet another embodiment, the HRS splicevariant polypeptide is a HRS fragment that lacks a functionalaminoacylation domain, e.g., amino acid residues 54-398 of the humanfull length HRS protein. In a more particular embodiment, the HRS splicevariant polypeptide comprises at least the WHEP domain and the anticodonbinding domain but lacks a functional aminoacylation domain.

In a more specific embodiment, the HRS polypeptide of the inventioncomprises a sequence set forth in SEQ ID NOs: 6, 9 or 11, or is acontiguous fragment of a polypeptide set forth in SEQ ID NOs: 6, 9 or11. Illustratively, the fragments may be of essentially any length,provided they retain at least one non-canonical biological activity ofinterest. For example, as further described herein, such a fragment maycomprise at least about 5, 10, 15, 20, 25, 50, 75 or 80, or more,contiguous amino acid residues of SEQ ID NOs: 6, 9 or 11.

In further embodiments of the invention, a HRS polypeptide comprises anactive variant (i.e., retains at least one non-canonical biologicalactivity of interest) of a sequence set forth in SEQ ID NOs: 6, 9 or 11.In a more specific embodiment, the active variant is a polypeptidehaving at least 70%, 80%, 90%, 95% or 99% identity along its length to asequence set forth in SEQ ID NOs: 6, 9 or 11.

In another particular embodiment, the HRS polypeptide of the inventionis not a polypeptide consisting of residues 1-48 of the full lengthhuman HRS protein.

According to another aspect of the invention, there are provided fusionproteins comprising at least one HRS polypeptide as described herein anda heterologous fusion partner.

According to another aspect of the invention, there are providedisolated polynucleotides encoding the polypeptides and fusion proteinsas described herein, as well as expression vectors comprising suchpolynucleotides, and host cell comprising such expression vectors. Alsoincluded are oligonucleotides that specifically hybridize to an HRSpolynucleotide, such as the polynucleotides of SEQ ID NOS:5, 8, or 10.In certain embodiments, the oligonucleotide is a primer, a probe, or anantisense oligonucleotide. Other embodiments relate to RNAi agents thattarget an HRS polynucleotide. In certain embodiments, theoligonucleotides or RNAi agents specifically hybridize to or otherwisetarget a splice junction that is unique to the HRS splice variant.

According to another aspect of the invention, there are provided bindingagents (e.g., antibodies and antigen-binding fragments thereof) thathave binding specificity for a HRS splice variant polypeptide of theinvention (e.g., SEQ ID NOs: 6, 9, or 11), or one of its cellularbinding partners. In certain embodiments, the binding agent is anantibody, an antigen-binding fragment thereof, a peptide, a peptidemimetic, a small molecule, or an aptamer. In some embodiments, thebinding agent antagonizes a non-canonical activity of the HRSpolypeptide. In other embodiments, the binding agent agonizes anon-canonical activity of the HRS polypeptide.

According to yet another aspect of the invention, there are providedcompositions, e.g., pharmaceutical compositions, comprisingphysiologically acceptable carriers and at least one of the isolatedpolypeptides, fusion proteins, antibodies, isolated polynucleotides,expression vectors, host cells, etc., of the invention, as describedherein.

Also included are methods of determining presence or levels of apolynucleotide sequence of a HRS splice variant in a sample, comprisingcontacting the sample with one or more oligonucleotides thatspecifically hybridize to an HRS splice variant as set forth SEQ IDNOS:5, 8, or 10, detecting the presence or absence of theoligonucleotides in the sample, and thereby determining the presence orlevels of the polynucleotide sequence of the HRS splice variant.

Also provided are methods of determining presence or levels of apolynucleotide sequence of a HRS splice variant in a sample, comprisingcontacting the sample with at least two oligonucleotides thatspecifically amplify an HRS splice variant as set forth in SEQ ID NOS:5,8, or 10, performing an amplification reaction, detecting the presenceor absence of an amplified product, and thereby determining presence orlevels of the polynucleotide sequence of the HRS splice variant. In someembodiments, the oligonucleotide(s) specifically hybridize to orspecifically amplify a splice junction that is unique to the HRS splicevariant. Certain embodiments include comparing the presence or levels ofthe HRS splice variant to a control sample or a predetermined value.Specific embodiments include characterizing the state of the sample todistinguish it from the control. In particular embodiments, the sampleand control comprise a cell or tissue, and the method comprisesdistinguishing between cells or tissues of different species, cells ofdifferent tissues or organs, cells at different cellular developmentalstates, cells at different cellular differentiation states, or healthyand diseased cells.

Also included are methods of identifying a compound that specificallybinds to a HRS splice variant polypeptide as set forth in SEQ ID NOS:6,9, or 11, or one or more of its cellular binding partners, comprising a)combining the HRS polypeptide or its cellular binding partner or bothwith at least one test compound under suitable conditions, and b)detecting binding of the HRS polypeptide or its cellular binding partneror both to the test compound, thereby identifying a compound thatspecifically binds to the HRS polypeptide or its cellular bindingpartner or both. In certain embodiments, the test compound is apolypeptide or peptide, an antibody or antigen-binding fragment thereof,a peptide mimetic, or a small molecule. In some embodiments, the testcompound agonizes a non-canonical biological activity of the HRSpolypeptide or its cellular binding partner. In other embodiments, thetest compound antagonizes a non-canonical biological activity of the HRSpolypeptide or its cellular binding partner. Also included are compoundsidentified by any of the methods provided herein.

Also provided by the present invention, in other aspects, are methodsfor modulating a cellular activity by contacting a cell or tissue with acomposition of the invention, as described herein. For example, incertain embodiments, the cellular activity to be modulated is selectedfrom the group consisting of cytokine production, cell proliferation,apoptosis, cell signaling, cellular metabolism, angiogenesis, cellmigration, cell binding, and the like. In a specific embodiment, themethod is a method for modulating cytokine production. In a morespecific embodiment, the method is a method for modulating IL-2production and/or secretion. In some embodiments, the method is a methodfor modulating TNF-α production and/or secretion. In other embodiments,the method is a method for modulating MIP1-α production and/orsecretion.

In certain embodiments, the cellular activity is cytokine receptoractivity. In specific embodiments, the cytokine receptor is CCR1. Incertain embodiments, the cellular activity is cell migration. Someembodiments include reducing cell migration of monocytes. In certainembodiments, the cellular activity is cell signaling through Toll-likereceptors (TLR)s.

In other aspects, the present invention provides methods for treating adisease, disorder or other condition in a subject in need thereof byadministering a composition according to the present invention. By wayof illustration, such diseases, disorders or conditions may include, butare not limited to, cancer, inflammatory disease, immune disease(including autoimmune disease) and/or conditions associated withabnormal angiogenesis.

In certain embodiments, the condition is a neurological condition. Inspecific embodiments, the neurological condition is associated with6-hydroxydopamine (6-OHDA)-induced neuron death. In certain embodiments,the condition is an inflammatory disease. In specific embodiments, theinflammatory disease is arthritic gout or inflammatory bowel disease.

In still other aspects, the polypeptides, antibodies and/or othercompositions of the present invention may be used in essentially anytype of screening assay known and available in the art. For example,compositions of the invention (e.g., polypeptides, polynucleotidesand/or antibodies) may be used in conjunction with essentially any knownscreening methodology in order to identify suitable cell types and/ordisease conditions amenable to treatment according to the presentinvention. In other examples, compositions of the invention (e.g.,polypeptides, polynucleotides and/or antibodies) may be used inconjunction with known screening methodologies in order to identifybinding partners, competitive inhibitors and/or other cellular effectorsthat mediate or modulate, either directly or indirectly, thenon-canonical activities of the compositions herein. For example, in aparticular embodiment, a screening method is provided for identifyingtest compounds as inhibitors, or alternatively, potentiators, of aninteraction between a composition of the invention and one or more ofits binding partners, cellular effectors and/or cell types subject tomodulation. This may include, for example, steps of forming a reactionmixture including: (i) a composition of the invention, (ii) a bindingpartner, cellular effector and/or cell type known to be modulated bysaid composition, and (iii) a test compound; and detecting interactionof the test compound with the binding partner, cellular effector and/orcell type. A statistically significant change (potentiation orinhibition) in activity or modulation in the presence of the testcompound, relative to the interaction in the absence of the testcompound, indicates a potential agonist (mimetic or potentiator) orantagonist (inhibitor) of activity.

BRIEF DESCRIPTION OF SEQUENCE IDENTIFIERS

SEQ ID NO: 1 is a primer sequence (HRS-BPF).

SEQ ID NO: 2 is a primer sequence (HRS-P1R).

SEQ ID NO: 3 is the nucleic acid sequence of the HRS gene (NM_002109.3).

SEQ ID NO: 4 is the amino acid sequence of the full length HRS protein(NP_002100.2)

SEQ ID NO: 5 is a nucleic acid coding sequence of the HRS-SV9 splicevariant.

SEQ ID NO: 6 is the amino acid sequence of the HRS-SV9 splice variantpolypeptide encoded by SEQ ID NO:5.

SEQ ID NO: 7 is a primer sequence (HRS-3′-UTR).

SEQ ID NO: 8 is a nucleic acid coding sequence of the HRS-SV11 splicevariant.

SEQ ID NO: 9 is the amino acid sequence of the HRS-SV11 splice variantpolypeptide encoded by SEQ ID NO:8.

SEQ ID NO:10 is a nucleic acid coding sequence of the HRS-SV14 splicevariant.

SEQ ID NO:11 is the amino acid sequence of the HRS-SV14 splice variantpolypeptide encoded by SEQ ID NO:10.

SEQ ID NO:12 is a primer sequence (hsH1-E2F1).

SEQ ID NO:13 is a primer sequence (hsH1-E13R1).

SEQ ID NO:14 is a primer sequence (rnH1-E02F1).

SEQ ID NO:15 is a primer sequence (rnH1-E12J13R2).

SEQ ID NO:16 is amino acids 112-171 of HRS from Pongo abelii(orangutan).

SEQ ID NO:17 is amino acids 112-171 of bovine HRS.

SEQ ID NO:18 is amino acids 112-171 of mouse HRS.

SEQ ID NO:19 is amino acids 112-171 of HRS from Mesocricetus auratus(golden hamster).

SEQ ID NO:20 is amino acids 112-166 of HRS from Fugu rubripes (Japanesepuffer fish).

SEQ ID NO:21 is amino acids 112-167 of HRS from Caenorhabditis elegans.

SEQ ID NO:22 is amino acids 112-169 of HRS from Dictyosteliumdiscoideum.

SEQ ID NO:23 is amino acids 112-167 of HRS from Oryza sativa subsp.japonica (rice).

SEQ ID NO:24 is a portion of the coding sequence of exons 3 and 4 ofhuman HRS.

SEQ ID NO:25 is a portion of the amino acid sequence of exons 3 and 4 ofhuman HRS.

SEQ ID NO:26 is a portion of the coding sequence of exons 3 and 4 ofhuman HRS.

SEQ ID NO:27 is a portion of the coding sequence of exons 10 and 11 ofhuman HRS.

SEQ ID NO:28 is a portion of the amino acid sequence of exons 10 and 11of human HRS.

SEQ ID NO:29 is amino acids 112-171 of HRS-SV11.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B show the identification of the HRS-SV9 splice variant from ahuman skeletal muscle library. FIG. 1A shows an illustration of an mRNAtranscript of the HRS gene and primer positions for PCR reactions (BPF:forward primer, P4R: reverse primer). FIG. 1B shows a gel photo of PCRreaction products from human skeletal muscle, IMR32 cells and HEK293Tcells. The upper arrow points to a DNA fragment amplified from theHRS-SV9 transcript. The lower arrow points to a DNA fragment amplifiedfrom HRS reference sequence.

FIGS. 2A-C show the identification of the HRS-SV11 splice variant froman IMR32 cell library and in samples from the human brain. FIG. 2A showsan illustration of an mRNA transcript of the HRS gene and primerpositions for PCR reactions (BPF: forward primer, HRS-3′UTR: reverseprimer). FIG. 2B shows a gel photo of PCR reaction products from IMR32cells. The lower arrow points to a DNA fragment amplified from theHRS-SV11 transcript. The upper arrow points to a DNA fragment amplifiedfrom the HRS reference sequence. FIG. 2C shows that the HRS-SV11 splicevariant transcript was identified in human brain tissue.

FIGS. 3A-D illustrate mRNA transcripts and protein sequences of wildtype, full length HRS (HRS ref), HRS-SV9, HRS-SV11, and HRS-SV14. FIG.3A shows an illustration of mRNA transcripts showing that HRS-SV9 has aninsertion from Intron 2 and HRS-SV11 has a deletion from Exon 3 to Exon10. FIG. 3B shows protein structural information encoded by the mRNAtranscripts, showing that HRS-SV9 has only the first 60 amino acids ofHRS, including the intact WHEP domain, whereas HRS-SV11 has a deletionof the whole aminoacylation domain, leaving only the WHEP and anticodondomains. FIGS. 3C and 3D show the nucleic acid coding sequences (SEQ IDNOs: 5, 8, and 10) and encoded protein sequences (SEQ ID NOs: 6, 9, and11) for HRS-SV9, HRS-SV11, and HRS-SV14, respectively.

FIGS. 4A-B show immunoblot results using anti-HRS antibodies in rattibialis muscle (Tib), soleus muscle (Sol), C2C12 myotubes (C2), adultrat brain (Br) and HEK293T cells (293T). FIG. 4A shows an immunoblotwith the N-terminal HRS antibody (against amino acids 1-97). Thereference band is shown by the upper arrow of FIG. 4A. The lower arrowof FIG. 4A points to a band whose size is consistent with predicted sizeof the HRS-SV11 splice variant polypeptide. FIG. 4B shows an immunoblotwith the C-terminal HRS antibody (against 50-200 amino acids near theC-terminus). The upper arrow points to the reference protein, whilelower arrow points to a band with similar size as seen with theN-terminal antibody.

FIGS. 5A-C show results of immunoprecipitation experiments with anN-terminal HRS monoclonal antibody (raised against amino acids 1-97 ofwild-type human HRS protein). FIG. 5A shows results for HEK293T cells,C2C12 myoblasts (MB) and C2C12 myotubes (MT); the lower arrow points toa band having a size that is consistent with the predicted size of theHRS-SV9 splice variant polypeptide. FIGS. 5B-C show the results fortotal cell lysate of IMR32 and HEK293T cells that over-express amyc-tagged HRS-SV11; the cells were immunoblotted with either theN-terminal HRS mAb (FIG. 5B), or a polyclonal HRS antibody (raisedagainst the C-terminus of wild type human HRS protein) (FIG. 5C). Aprotein band, which migrated slightly faster than myc-tagged HRS-SV11protein, was detected in IMR32 cell lysate by both antibodies (lowerarrows in B and C) and could be the HRS-SV11 protein.

FIGS. 6A-C demonstrate the secretion of HRS, HRS-SV9 and HRS-SV11following recombinant production in HEK293T cells. Wild type, fulllength HRS (HRS-Ref), HRS-SV9 and HRS-SV11 were forcefully expressed inHEK293T cells. FIG. 6A, upper panel, shows arrows pointing tooverexpressed proteins in total cell lysates (TCL). FIG. 6B, lowerpanel, shows that in media fractions all three proteins were detected.HRS-Ref was probed with anti-Myc antibody, while HRS-SV9 and HRS-SV11were probed with anti-HRS (N-terminus) antibody. A tubulin blot in TCLshowed equal loading, while a tubulin blot in the media fractiondemonstrated leaky control. FIG. 6C shows that EGFP was not secretedwhen over-expressed in HEK293T cells, as indicated by the absence of anEGFP band (˜35 kDa) in the media fraction.

FIG. 7 shows that recombinant HRS-SV9 and HRS-SV11 splice variantpolypeptides enhance IL-2 secretion in activated Jurkat T cells. Cellswere treated with PMA (25 ng/ml) plus ionomycin (250 ng/ml) with orwithout HRS-SV9 or HRS-SV11, and media was analyzed 48 hours later byELISA.

FIG. 8 shows that HRS-SV9 stimulated PBMCs to release TNFα into theculture supernatant. LPS was used as a positive control.

FIGS. 9A-D show that HRS-SV11 protected cultured cortical neuron andPC12 cells against neurotoxin 6-OHDA. FIG. 9A shows that pre-treatingrat cortical neurons with HRS-SV11 significantly reduced cortical neurondeath induced by 25 μM 6-OHDA as measurement of cell viability by MTTassay. HRS-SV11 also effectively protected PC12 cells from 6-OHDA (200μM)-induced cell death, as shown by MTT assay (FIG. 9B) and LDH assay(FIG. 9C). As shown in FIG. 9D, HRS-SV11's protective effect was anacute effect, with short time pre-treatment achieved similar protectiveeffect as 24 hrs pre-treatment. Data are means±S.D. from three separateexperiments. Tert-butylhydroquinone (tBHQ) and Triton X-100 (in shortTriton) (2%) served as positive controls for MTT and LDH assays,respectively (B, C). *p<0.05, **p<0.01.

FIGS. 10A-F show that HRS-SV11 did not protect neurons from amyloidbeta, monosodium glutamate (MSG), and MPP+-induced toxicity.

FIG. 10A shows the results of incubation with β-amyloid (1-42) (Aβ₄₂□)aggregates for 24 hrs, which induced cortical neuron death in adose-dependent manner. FIG. 10B shows that pre-treating cortical neuronsfor 24 hrs with HRS-SV11, from 1 nM to 1 μM, had no protective effect asmeasured by MTT assay. FIG. 10C shows monosodium glutamate (MSG) inducedcortical neuron death in a dose-dependent manner as measurement of LDHrelease, and FIG. 10D shows that HRS-SV11 had no beneficial effectagainst monosodium glutamate-induced toxicity; memantine at 10 μMsignificantly prevented neuron death, serving as a positive control.FIG. 10E shows that MPP+ induced PC12 cell death in a dose-dependentmanner as measured by MTT assay, and FIG. 10F shows that HRS-SV11 didnot protect PC12 cells from MPP+ stress after 24 hrs pre-treatment. Dataare means±S.D. from three separate experiments.

FIGS. 11A-E show that HRS-SV11 does not utilize and extracellularmechanism to protect neurons from 6-OHDA. As shown in FIG. 11A, additionof HRS-SV11 did not suppress p-quinone's accumulation, while vitamin C,a known anti-oxidant, did suppress its accumulation. FIG. 11B shows thathydrogen peroxide (H₂O₂) induced cortical neuron death in adose-dependent manner, and FIG. 11C shows that pre-treating withHRS-SV11 did not protect these neurons from death. As shown in FIG. 11D,pre-treating cortical neurons with HRS-SV11 reduced neuron death upon6-OHDA challenge, but co-application of HRS-SV11 with 6-OHDA had noprotective effect. Before 6-OHDA application, washing and refreshingmedia after HRS-SV11 pre-treatment did not affect HRS-SV11's protectiveeffect (see FIG. 11E). Data are means±S.D. from three separateexperiments. *p<0.05, **p<0.01.

FIGS. 12A-D show that HRS-SV11 prevented DNA fragmentation induced by6-OHDA in PC12 cells. Apoptosis of PC12 cells was examined by Hoechst33258 staining. FIG. 12A shows PC12 cells treated with buffer controlalone; FIG. 12B shows treatment with 6-OHDA (200 μM) for 8 h; and FIG.12C shows pre-treatment with HRS-SV11 (500 nM) for 24 h followed by 8 hchallenge with 6-OHDA (200 μM). As shown in FIG. 12D, numbers ofapoptotic cells were counted (distinguished by presence of fragmentednuclei), demonstrating that pre-treatment with 1 μM HRS-SV11 greatlyreduced the number of apoptotic cells.

FIGS. 13A-C show that mutation of cysteine (Cys) residues in theC-terminus of HRS-SV11 abolished the protective function. FIG. 13A showsthat HRS-SV11 (SEQ ID NO:29) contains three Cys residues (C117, C169 andC171). Among them, C117 and C171 (arrows, FIG. 13A) are highly conservedacross species, as shown by the alignment of amino acids 112 to about171 of a variety of HRS sequences (SEQ ID NOS:16-24 and 29). As shown inFIG. 13B, the C2S (mutation of C169 and C171 into serine residue) anddeIC (deletion of last three amino acids, including C169 and C171)mutants were mostly monomer, while the C117S mutant was mostly dimer.The wild type HRS-SV11 had a peak in between monomer and dimer,indicating there was a very dynamic switch between these two forms. Asshown in FIG. 13C, mutations of C169 and C171 into serine residue(HRS-SV11_C2S) or deletion of last three amino acids, including C169 andC171 (HRS-SV11_delC) abolished HRS-SV11's neuroprotective function,suggesting critical role of these Cys. **p<0.01.

FIGS. 14A-D show that the inhibition of JAK2, JNK and p38 suppressed theneuroprotective effect of HRS-SV11. As shown in FIGS. 14A-B, HRS-SV11'sneuroprotective effect in PC12 cells was suppressed by co-inhibition ofJNK by SB202190 at 10 μM and p38 by SP600125 at 10 μM (FIG. 14A), and byinhibition of JAK2 by AG490 at 40 μM (FIG. 14B). FIG. 14B also showsthat the neuroprotective effect of HRS-SV11 was not suppressed by theinhibition of phospholipase C (PLC) by U73122, or MKK by arctigenin.FIGS. 14C-D show that similar observations were made with corticalneurons. Data are means±S.D. from three separate experiments. *p<0.05,**p<0.01.

FIGS. 15A-D show that HRS-SV11 bound to CCR5-expressing HEK293T cells,but not to CCR1-expressing or non-transfected cells. As in FIGS. 15A-B,HRS-SV11 did not bind to HEK293T cells (FIG. 15A), but bound toCCR5-expressing HEK293T cells (FIG. 15B). As shown in FIG. 15C, bindingto CCR-5 expressing cells was not affected by pre-treating cells withMet-RANTES. FIG. 15D shows that binding was specific to CCR5, since nobinding was observed on CCR1-expressing cells. Control cells wereincubated with FITC-His antibody only.

FIGS. 16A-D show identification of the HRS-SV14 splice variant andneuroprotection of HRS-SV14. FIG. 16A shows that the identification of anew splicing variant of human HRS in human fetus brain cDNA (arrow). Asshown in FIGS. 16A-B, cloning and sequencing revealed that this HRSsplicing variant results from skipping of Exon 4 to Exon 10 in thewild-type HRS transcript (SEQ ID NOS:24-28 are in order from the upperto the lower part of FIG. 16B). On the protein level, HRS-SV14 proteinwas predicted to contain the WHEP domain, anticodon binding domain, andthe first motif of the aminoacylation domain (see FIG. 16C). FIG. 16Dshows that HRS-SV14, when pre-treated for 24 hr, protected PC12 cellsfrom 6-OHDA-induced neuron death; and the effect was comparable toHRS-SV11. Data are means±S.D. from three separate experiments. *p<0.05.

FIG. 17 shows the detection of HRS-SV11 and SV14 transcripts indifferent tissues and cell lines. This figure shows the electrophoresisof PCR products flanking Exon 2 and Exon 12 of HARS from cDNA of humanadult brain, fetus brain, lung, skeletal muscle tissues and IMR32,Jurkat and THP-1 cells. HRS-SV11, as indicated by the horizontal arrow,was present in all the samples, except fetus brain. HRS-SV14, asindicated by angled arrows, was detected in human fetus brain, lung,skeletal muscle, Jurkat and THP-1 cells.

FIGS. 18A-C show that the protective effect of HRS-SV11 recombinantprotein was not from non-protein contaminants. FIG. 18A shows coomassieblue staining of recombinant HRS-SV11 protein preparation with orwithout proteinase K digestion; no visible protein band was detected inproteinase K-treated HRS-SV11 preparation, as compared to untreatedHRS-SV11 preparation (arrowhead indicates HRS-SV11 recombinant protein).As shown in FIGS. 18B-C, pre-treating cortical neurons (FIG. 18B) andPC12 cells (FIG. 18C) with proteinase K-digested HRS-SV11 abolished itsprotective effect against 6-OHDA. tBHQ served as a positive control. NSstands for no significance. Data are means±S.D. from three separateexperiments. *p<0.05, **p<0.01.

FIG. 19 shows that SV9 inhibits the migration of monocytes (THP-1 cells)towards CCL5.

FIG. 20 shows that SV9 inhibits CCR1-mediated migration of THP-1 cellstowards CCL-23.

FIG. 21 shows activation of macrophage TLRs by SV9. LPS is a positivecontrol

FIGS. 22A and 22B show that SV9 activates both TLR2 (22A) and TLR4(22B), but preferentially activates TLR4.

FIG. 23 shows that SV9 stimulates MIP-1α secretion in monocytes (THP-1).

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of molecular biologyand recombinant DNA techniques within the skill of the art, many ofwhich are described below for the purpose of illustration. Suchtechniques are explained fully in the literature. See, e.g., Sambrook,et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989);Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNACloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., 1985); Transcription andTranslation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R.Freshney, ed., 1986); A Practical Guide to Molecular Cloning (B. Perbal,ed., 1984).

All publications, patents and patent applications cited herein arehereby incorporated by reference in their entirety.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise. By way of example, “an element” means oneelement or more than one element.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

An “agonist” refers to a molecule that intensifies or mimics thenon-canonical biological activity of an HRS. Agonists may includeproteins, nucleic acids, carbohydrates, small molecules, or any othercompound or composition that modulates the activity of an HRS either bydirectly interacting with the HRS or its binding partner, or by actingon components of the biological pathway in which the HRS participates.Included are partial and full agonists.

The term “antagonist” refers to a molecule that inhibits or attenuatesthe non-canonical biological activity of an HRS. Antagonists may includeproteins such as antibodies, nucleic acids, carbohydrates, smallmolecules, or any other compound or composition that modulates theactivity of an HRS or its binding partner, either by directlyinteracting with the HRS or its binding partner or by acting oncomponents of the biological pathway in which the HRS participates.Included are partial and full antagonists.

By “coding sequence” is meant any nucleic acid sequence that contributesto the code for the polypeptide product of a gene. By contrast, the term“noncoding sequence” refers to any nucleic acid sequence that does notcontribute to the code for the polypeptide product of a gene.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises,” and “comprising” will be understoodto imply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of.” Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present. By “consisting essentially of” is meantincluding any elements listed after the phrase, and limited to otherelements that do not interfere with or contribute to the activity oraction specified in the disclosure for the listed elements. Thus, thephrase “consisting essentially of” indicates that the listed elementsare required or mandatory, but that other elements are optional and mayor may not be present depending upon whether or not they materiallyaffect the activity or action of the listed elements.

As used herein, the terms “function” and “functional” and the like referto a biological, enzymatic, or therapeutic function.

By “gene” is meant a unit of inheritance that occupies a specific locuson a chromosome and consists of transcriptional and/or translationalregulatory sequences and/or a coding region and/or non-translatedsequences (i.e., introns, 5′ and 3′ untranslated sequences).

“Homology” refers to the percentage number of amino acids that areidentical or constitute conservative substitutions. Homology may bedetermined using sequence comparison programs such as GAP (Deveraux etal., 1984, Nucleic Acids Research 12, 387-395), which is incorporatedherein by reference. In this way sequences of a similar or substantiallydifferent length to those cited herein could be compared by insertion ofgaps into the alignment, such gaps being determined, for example, by thecomparison algorithm used by GAP.

The term “host cell” includes an individual cell or cell culture thatcan be or has been a recipient of any recombinant vector(s) or isolatedpolynucleotide of the invention. Host cells include progeny of a singlehost cell, and the progeny may not necessarily be completely identical(in morphology or in total DNA complement) to the original parent celldue to natural, accidental, or deliberate mutation and/or change. A hostcell includes cells transfected or infected in vivo or in vitro with arecombinant vector or a polynucleotide of the invention. A host cellwhich comprises a recombinant vector of the invention is a recombinanthost cell.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Forexample, an “isolated polynucleotide,” as used herein, includes apolynucleotide that has been purified from the sequences that flank itin its naturally-occurring state, e.g., a DNA fragment which has beenremoved from the sequences that are normally adjacent to the fragment.Alternatively, an “isolated peptide” or an “isolated polypeptide” andthe like, as used herein, includes the in vitro isolation and/orpurification of a peptide or polypeptide molecule from its naturalcellular environment, and from association with other components of thecell; i.e., it is not significantly associated with in vivo substances.

The term “mRNA” or sometimes refer by “mRNA transcripts” as used herein,include, but not limited to pre-mRNA transcript(s), transcriptprocessing intermediates, mature mRNA(s) ready for translation andtranscripts of the gene or genes, or nucleic acids derived from the mRNAtranscript(s). Transcript processing may include splicing, editing anddegradation. As used herein, a nucleic acid derived from an mRNAtranscript refers to a nucleic acid for whose synthesis the mRNAtranscript or a subsequence thereof has ultimately served as a template.A cDNA reverse transcribed from an mRNA, an RNA transcribed from thatcDNA, a DNA amplified from the cDNA, an RNA transcribed from theamplified DNA, etc., are all derived from the mRNA transcript anddetection of such derived products is indicative of the presence and/orabundance of the original transcript in a sample. Thus, mRNA derivedsamples include, but are not limited to, mRNA transcripts of the gene orgenes, cDNA reverse transcribed from the mRNA, cRNA transcribed from thecDNA, DNA amplified from the genes, RNA transcribed from amplified DNA,and the like.

“Non-canonical” activity as used herein, refers generally to an activitypossessed by an HRS polypeptide of the invention that is other thanaminoacylation and, more specifically, other than the addition of itscognate amino acid onto its cognate tRNA molecule. Non-limiting examplesof non-canonical activities include RNA-binding, amino acid-binding,modulation of cell proliferation, modulation of cell migration,modulation of cell differentiation (e.g., hematopoiesis), modulation ofapoptosis or other forms of cell death, modulation of cell signaling,modulation of angiogenesis, modulation of cell binding, modulation ofcellular metabolism, modulation of cytokine production or activity,modulation of cytokine receptor activity, modulation of inflammation,and the like.

The term “modulating” includes “increasing” or “stimulating,” as well as“decreasing” or “reducing,” typically in a statistically significant ora physiologically significant amount as compared to a control. An“increased” or “enhanced” amount is typically a “statisticallysignificant” amount, and may include an increase that is 1.1, 1.2, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times)(including all integers and decimal points in between and above 1, e.g.,1.5, 1.6, 1.7. 1.8, etc.) the amount produced by no composition (theabsence of an agent or compound) or a control composition. A “decreased”or reduced amount is typically a “statistically significant” amount, andmay include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease in the amountproduced by no composition (the absence of an agent or compound) or acontrol composition, including all integers in between. Other examplesof “statistically significant” amounts are described herein.

By “obtained from” is meant that a sample such as, for example, apolynucleotide extract or polypeptide extract is isolated from, orderived from, a particular source of the subject. For example, theextract can be obtained from a tissue or a biological fluid isolateddirectly from the subject. “Derived” or “obtained from” can also referto the source of a polypeptide or polynucleotide sequence. For instance,an HRS sequence of the present invention may be “derived” from thesequence information of an HRS proteolytic fragment or HRS splicevariant, or a portion thereof, whether naturally-occurring orartificially generated, and may thus comprise, consist essentially of,or consist of that sequence.

The recitations “sequence identity” or, for example, comprising a“sequence 50% identical to,” as used herein, refer to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity.

A “splice junction” as used herein includes the region in a mature mRNAtranscript or the encoded polypeptide where the 3′ end of a first exonjoins with the 5′ end of a second exon. The size of the region may vary,and may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100 or more (including all integers in between) nucleotide or amino acidresidues on either side of the exact residues where the 3′ end of oneexon joins with the 5′ end of another exon. An “exon” refers to anucleic acid sequence that is represented in the mature form of an RNAmolecule after either portions of a precursor RNA (introns) have beenremoved by cis-splicing or two or more precursor RNA molecules have beenligated by trans-splicing. The mature RNA molecule can be a messengerRNA or a functional form of a non-coding RNA such as rRNA or tRNA.Depending on the context, an exon can refer to the sequence in the DNAor its RNA transcript. An “intron” refers to a non-coding nucleic acidregion within a gene, which is not translated into a protein. Non-codingintronic sections are transcribed to precursor mRNA (pre-mRNA) and someother RNAs (such as long noncoding RNAs), and subsequently removed bysplicing during the processing to mature RNA.

A “splice variant” refers to a mature mRNA and its encoded protein thatare produced by alternative splicing, a process by which the exons ofthe RNA (a primary gene transcript or pre-mRNA) are reconnected inmultiple ways during RNA splicing. The resulting different mRNAs may betranslated into different protein isoforms, allowing a single gene tocode for multiple proteins.

A “subject,” as used herein, includes any animal that exhibits asymptom, or is at risk for exhibiting a symptom, which can be treated ordiagnosed with an HRS polynucleotide or polypeptide of the invention.Suitable subjects (patients) include laboratory animals (such as mouse,rat, rabbit, or guinea pig), farm animals, and domestic animals or pets(such as a cat or dog). Non-human primates and, preferably, humanpatients, are included.

“Treatment” or “treating,” as used herein, includes any desirable effecton the symptoms or pathology of a disease or condition that can beeffected by the non-canonical activities of an HRS polynucleotide orpolypeptide, as described herein, and may include even minimal changesor improvements in one or more measurable markers of the disease orcondition being treated. Also included are treatments that relate tonon-HRS therapies, in which an HRS sequence described herein provides aclinical marker of treatment. “Treatment” or “treating” does notnecessarily indicate complete eradication or cure of the disease orcondition, or associated symptoms thereof. The subject receiving thistreatment is any subject in need thereof. Exemplary markers of clinicalimprovement will be apparent to persons skilled in the art.

By “vector” or “nucleic acid construct” is meant a polynucleotidemolecule, preferably a DNA molecule derived, for example, from aplasmid, bacteriophage, yeast or virus, into which a polynucleotide canbe inserted or cloned. A vector preferably contains one or more uniquerestriction sites and can be capable of autonomous replication in adefined host cell including a target cell or tissue or a progenitor cellor tissue thereof, or be integrable with the genome of the defined hostsuch that the cloned sequence is reproducible. Accordingly, the vectorcan be an autonomously replicating vector, i.e., a vector that exists asan extra-chromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a linear or closed circular plasmid, anextra-chromosomal element, a mini-chromosome, or an artificialchromosome. The vector can contain any means for assuringself-replication. Alternatively, the vector can be one which, whenintroduced into the host cell, is integrated into the genome andreplicated together with the chromosome(s) into which it has beenintegrated.

The terms “wild-type” and “naturally occurring” are used interchangeablyto refer to a gene or gene product that has the characteristics of thatgene or gene product when isolated from a naturally occurring source. Awild-type gene or gene product (e.g., a polypeptide) is that which ismost frequently observed in a population and is thus arbitrarilydesigned the “normal” or “wild-type” form of the gene.

HRS Splice Variant Polypeptides

As noted above, according to one aspect of the invention, there areprovided HRS “splice variant” polypeptides having non-canonicalactivities of therapeutic relevance, as well as compositions comprisingthe same.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues andto variants and synthetic analogues of the same. Thus, these terms applyto amino acid polymers in which one or more amino acid residues aresynthetic non-naturally occurring amino acids, such as a chemicalanalogue of a corresponding naturally occurring amino acid, as well asto naturally-occurring amino acid polymers.

Polypeptides are not limited to a specific length, but, in the contextof the present invention, typically represent a fragment of a fulllength protein, and may include post-translational modifications, forexample, glycosylations, acetylations, phosphorylations and the like, aswell as other modifications known in the art, both naturally occurringand non-naturally occurring. Polypeptides and proteins of the inventionmay be prepared using any of a variety of well known recombinant and/orsynthetic techniques, illustrative examples of which are furtherdiscussed below.

The recitation “polypeptide variant” refers to polypeptides that aredistinguished from a reference HRS splice variant polypeptide (e.g., SEQID NOS:6, 9, 11) by the addition, deletion, and/or substitution of atleast one amino acid residue, and which typically retain thenon-canonical activity of the reference HRS splice variant polypeptide.In certain embodiments, a polypeptide variant is distinguished from areference polypeptide by one or more substitutions, which may beconservative or non-conservative, as described herein and known in theart. In certain embodiments, the polypeptide variant comprisesconservative substitutions and, in this regard, it is well understood inthe art that some amino acids may be changed to others with broadlysimilar properties without changing the nature of the activity of thepolypeptide.

In certain embodiments, a polypeptide variant includes an amino acidsequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or more sequence identity orsimilarity to a corresponding sequence of an HRS reference polypeptide,as described herein, and retains the non-canonical activity of thatreference polypeptide. Also included are sequences differing from thereference HRS sequences by the addition, deletion, or substitution of 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more amino acidsbut which retain the properties of the reference HRS polypeptide. Inother embodiments, variant polypeptides differ from the correspondingHRS reference sequences by at least 1% but less than 20%, 15%, 10% or 5%of the residues. (If this comparison requires alignment, the sequencesshould be aligned for maximum similarity. “Looped” out sequences fromdeletions or insertions, or mismatches, are considered differences.) Thedifferences are, suitably, differences or changes at a non-essentialresidue or a conservative substitution.

Also included are biologically active “fragments” of the HRS referencepolypeptides. Representative biologically active fragments generallyparticipate in an interaction, e.g., an intramolecular or aninter-molecular interaction. An inter-molecular interaction can be aspecific binding interaction or an enzymatic interaction. Aninter-molecular interaction can be between an HRS polypeptide and acellular binding partner, such as a cellular receptor or other hostmolecule that participates in the non-canonical activity of the HRSpolypeptide.

Typically, biologically active fragments comprise a domain or motif withat least one activity of an HRS reference polypeptide and may includeone or more (and in some cases all) of the various active domains, andinclude fragments having a non-canonical activity. In some cases,biologically active fragments of an HRS polypeptide have a biologicalactivity that is unique to the particular, truncated fragment, such thatthe full-length HRS polypeptide may not have that activity. In certaincases, the biological activity may be revealed by separating thebiologically active HRS polypeptide fragment from the other full-lengthHRS polypeptide sequences, or by altering certain residues of thefull-length HRS wild-type polypeptide sequence to unmask thebiologically active domains.

For example, in one illustrative embodiment, the HRS splice variantpolypeptide is a HRS fragment comprising at least the WHEP domain ofHRS, or an active fragment or variant thereof. In another illustrativeembodiment, the polypeptide is a HRS fragment comprising at least theanticodon binding domain of HRS, or an active fragment or variantthereof. In yet another illustrative embodiment, the polypeptide is aHRS fragment comprising at least the WHEP domain and the anticodonbinding domain of HRS. In still another illustrative embodiment, thepolypeptide is a HRS fragment lacking the aminoacylation domain andsubstantially devoid of aminoacylation activity.

In a more particular embodiment, the HRS splice variant polypeptide is apolypeptide comprising a sequence set forth in SEQ ID NOs:6, 9 or 11. Inanother embodiment, the HRS splice variant polypeptide is a polypeptidecomprising an active fragment of SEQ ID NOs: 6, 9 or 11 (i.e., afragment of SEQ ID NOs: 6, 9 or 11 that substantially retains at leastone non-canonical activity exhibited by SEQ ID NOs: 6, 9 or 11). Forexample, such a fragment may comprise at least about 5, 10, 15, 20, 25,or 50, or more, contiguous amino acid residues of SEQ ID NOs: 6, 9 or11, as well as all intermediate lengths. Intermediate lengths areintended to include all integers therebetween, for example, 6, 7, 8,etc., 51, 52, 53, etc. In addition, such a fragment may comprise atleast about 5, 10, 15, 20, 25, 50, 75, 100, 125, or 150, or more,contiguous amino acid residues of SEQ ID NO: 9, as well as allintermediate lengths.

A biologically active fragment of an HRS reference polypeptide can be apolypeptide fragment which is, for example, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,170 or more contiguous or non-contiguous amino acids, including allintegers in between, of the amino acid sequences set forth SEQ ID NOS:6,9, or 11. In certain embodiments, the C-terminal or N-terminal region ofany HRS reference polypeptide may be truncated by about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180 or more amino acids, includingall integers and ranges in between (e.g., 101, 102, 103, 104, 105), solong as the truncated HRS polypeptide retains the non-canonical activityof the reference HRS splice variant polypeptide. Suitably, thebiologically-active fragment has no less than about 1%, 10%, 25%, or 50%of an activity of the biologically-active (i.e., non-canonical activity)HRS reference polypeptide from which it is derived.

In other illustrative embodiments, a HRS fragment of SEQ ID NO: 6, 9 or11 may range in size from about 20-30, 20-40, 20-50, 20-60, 20-70,20-80, 20-90, 20-100, 20-125, 20-150 or 20-175 amino acids in length. Inother embodiments, the fragment will range in size from about 30-40,30-50, 30-60, 30-70, 30-80, 30-90, 30-100, 30-125, 30-150 or 30-175amino acids in length. In other embodiments, the fragment will range insize from about 40-50, 40-60, 40-70, 40-80, 40-90, 40-100, 40-125,40-150 or 40-175 amino acids in length. In still other illustrativeembodiments, the fragment will range in size from about 50-60, 50-70,50-80, 50-90, 50-100, 50-125, 50-150 or 50-175 amino acids in length.

In still other embodiments, the present invention provides activevariants of a HRS splice variant polypeptide (e.g., SEQ ID NOs: 6, 9 or11), wherein said variants substantially retain at least onenon-canonical activity exhibited by SEQ ID NOs: 6, 9 or 11. Certainillustrative variants of the sequence set forth in SEQ ID NOs: 6, 9 or11 include those having at least about 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (determined asdescribed below), along their lengths, to SEQ ID NOs: 6, 9 or 11.

A variant may differ from SEQ ID NOs: 6, 9 or 11 in one or moresubstitutions, deletions, additions and/or insertions. Such variants maybe naturally occurring or may be synthetically generated, for example,by modifying SEQ ID NOs: 6, 9 or 11 (or a polynucleotide encoding SEQ IDNOs: 6, 9 or 11) and evaluating their biological activity as describedherein using any of a number of techniques well known in the art.

In certain embodiments, a variant will contain conservativesubstitutions. A “conservative substitution” is one in which an aminoacid is substituted for another amino acid that has similar properties,such that one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. Modifications may be made in the structure ofthe polynucleotides and polypeptides of the present invention and stillobtain a functional molecule that encodes a variant or derivativepolypeptide with desirable characteristics. When it is desired to alterthe amino acid sequence of a polypeptide to create an equivalent, oreven an improved, variant of a HRS splice variant polypeptide of theinvention, one skilled in the art, for example, can change one or moreof the codons of the encoding DNA sequence according to Table 1.

Certain amino acids may be substituted for other amino acids in aprotein structure without appreciable loss of interactive bindingcapacity with structures such as, for example, antigen-binding regionsof antibodies or binding sites on substrate molecules. Since it is theinteractive capacity and nature of a protein that generally defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated that various changes may bemade in the polypeptide sequences of the disclosed compositions, orcorresponding DNA sequences which encode said polypeptides withoutappreciable loss of their desired utility or activity.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may also beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). For example, it is known that the relative hydropathiccharacter of the amino acid contributes to the secondary structure ofthe resultant protein, which in turn defines the interaction of theprotein with other molecules, for example, enzymes, substrates,receptors, DNA, antibodies, antigens, and the like. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte and Doolittle, 1982). These values are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine(0.4); threonine (0.7); serine (0.8); tryptophan (0.9); tyrosine (1.3);proline (1.6); histidine (3.2); glutamate (3.5); glutamine (3.5);aspartate (3.5); asparagine (3.5); lysine (3.9); and arginine (4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. Asdetailed in U.S. Pat. No. 4,554,101, the following hydrophilicity valueshave been assigned to amino acid residues: arginine (+3.0); lysine(+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalentprotein. In such changes, the substitution of amino acids whosehydrophilicity values are within ±2 is preferred, those within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions may be based on the relativesimilarity of the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Exemplarysubstitutions that take various of the foregoing characteristics intoconsideration are well known to those of skill in the art and include:arginine and lysine; glutamate and aspartate; serine and threonine;glutamine and asparagine; and valine, leucine and isoleucine.

Amino acid substitutions may further be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity and/orthe amphipathic nature of the residues. For example, negatively chargedamino acids include aspartic acid and glutamic acid; positively chargedamino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine,isoleucine and valine; glycine and alanine; asparagine and glutamine;and serine, threonine, phenylalanine and tyrosine. Other groups of aminoacids that may represent conservative changes include: (1) ala, pro,gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Avariant may also, or alternatively, contain non-conservative changes. Ina preferred embodiment, variant polypeptides differ from a nativesequence by substitution, deletion or addition of five amino acids orfewer. Variants may also (or alternatively) be modified by, for example,the deletion or addition of amino acids that have minimal influence onsecondary structure and hydropathic nature of the polypeptide.

Polypeptides may comprise a signal (or leader) sequence at theN-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

When comparing polypeptide sequences, two sequences are said to be“identical” if the sequence of amino acids in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted, forexample, using the Megalign program in the Lasergene suite ofbioinformatics software (DNASTAR, Inc., Madison, Wis.), using defaultparameters. This program embodies several alignment schemes described inthe following references: Dayhoff, M. O. (1978) A model of evolutionarychange in proteins—Matrices for detecting distant relationships. InDayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp.345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp.626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego,Calif.; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers,E. W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb.Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425;Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy—thePrinciples and Practice of Numerical Taxonomy, Freeman Press, SanFrancisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Nat'lAcad., Sci. USA 80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Nat'l Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nucl. AcidsRes. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. BLAST and BLAST 2.0 can be used, for example with theparameters described herein, to determine percent sequence identity forthe polynucleotides and polypeptides of the invention. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. For amino acid sequences, ascoring matrix can be used to calculate the cumulative score. Extensionof the word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment.

In one illustrative approach, the “percentage of sequence identity” isdetermined by comparing two optimally aligned sequences over a window ofcomparison of at least 20 positions, wherein the portion of thepolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the referencesequence (i.e., the window size) and multiplying the results by 100 toyield the percentage of sequence identity.

In certain embodiments of the invention, there are provided fusionpolypeptides, and polynucleotides encoding fusion polypeptides. Fusionpolypeptides refer to HRS splice variant polypeptides of the inventionthat have been covalently linked, either directly or indirectly via anamino acid linker, to one or more heterologous polypeptide sequences(fusion partners). The polypeptides forming the fusion protein aretypically linked C-terminus to N-terminus, although they can also belinked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminusto C-terminus. The polypeptides of the fusion protein can be in anyorder.

The fusion partner may be designed and included for essentially anydesired purpose provided they do not adversely affect the desiredactivity of the polypeptide. For example, in one embodiment, a fusionpartner comprises a sequence that assists in expressing the protein (anexpression enhancer) at higher yields than the native recombinantprotein. Other fusion partners may be selected so as to increase thesolubility of the protein or to enable the protein to be targeted todesired intracellular compartments or to be secreted outside the cell.Still further fusion partners may include affinity tags, whichfacilitate purification of the protein.

More generally, fusion to heterologous sequences, such as an Fcfragment, may be utilized to remove unwanted characteristics or toimprove the desired characteristics (e.g., pharmacokinetic properties)of an HRS polypeptide. For example, fusion to a heterologous sequencemay increase chemical stability, decrease immunogenicity, improve invivo targeting, and/or increase half-life in circulation of an HRSpolypeptide.

Fusion to heterologous sequences may also be used to createbi-functional fusion proteins, such as bi-functional proteins that arenot only possess a selected non-canonical activity through the HRSpolypeptide, but are also capable of modifying (i.e., stimulating orinhibiting) other pathways through the heterologous polypeptide.Examples of such pathways include, but are not limited to, variousimmune system-related pathways, such as innate or adaptive immuneactivation pathways, or cell-growth regulatory pathways, such asangiogenesis. In certain aspects, the heterologous polypeptide may actsynergistically with the HRS polypeptide to modulate a cellular pathwayin a subject. Examples of heterologous polypeptides that may be utilizedto create a bi-functional fusion protein include, but are not limitedto, thrombopoietin, cytokines (e.g., IL-11), chemokines, and varioushematopoietic growth factors, in addition to biologically activefragments and/or variants thereof.

Fusion polypeptides may generally be prepared using standard techniques.For example, DNA sequences encoding the polypeptide components of adesired fusion may be assembled separately, and ligated into anappropriate expression vector. The 3′ end of the DNA sequence encodingone polypeptide component is ligated, with or without a peptide linker,to the 5′ end of a DNA sequence encoding the second polypeptidecomponent so that the reading frames of the sequences are in phase. Thispermits translation into a single fusion polypeptide that retains thebiological activity of both component polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures, ifdesired. Such a peptide linker sequence is incorporated into the fusionprotein using standard techniques well known in the art. Certain peptidelinker sequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39 46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA83:8258 8262 (1986); U.S. Pat. Nos. 4,935,233 and 4,751,180. The linkersequence may generally be from 1 to about 50 amino acids in length.Linker sequences are not required when the first and second polypeptideshave non-essential N-terminal amino acid regions that can be used toseparate the functional domains and prevent steric interference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

In general, polypeptides and fusion polypeptides (as well as theirencoding polynucleotides) are isolated. An “isolated” polypeptide orpolynucleotide is one that is removed from its original environment. Forexample, a naturally-occurring protein is isolated if it is separatedfrom some or all of the coexisting materials in the natural system.Preferably, such polypeptides are at least about 90% pure, morepreferably at least about 95% pure and most preferably at least about99% pure. A polynucleotide is considered to be isolated if, for example,it is cloned into a vector that is not a part of the naturalenvironment.

In still other embodiments, a HRS splice variant polypeptide of theinvention may be part of a dimer. Dimers may include, for example,homodimers between two identical HRS polypeptides, heterodimers betweentwo different HRS polypeptides and/or heterodimers between a HRSpolypeptide and a heterologous polypeptide. The monomers and/or dimersmay be soluble and may be isolated or purified to homogeneity. Certainheterodimers, such as those between a HRS polypeptide and a heterologouspolypeptide, may be bi-functional.

Also included are monomers of HRS polypeptides, including isolated HRSmonomers that do not substantially dimerize with themselves(homodomerize) or with a second HRS polypeptide (heterodimerize),whether due to one or more substitutions, truncations, deletions,additions, chemical modifications, or a combination of thesealterations. In certain embodiments, monomeric HRS polypeptides possessbiological activities, including non-canonical activities, which are notpossessed by dimeric or multimeric HRS polypeptide complexes.

In other embodiments, a HRS polypeptide of the invention may be part ofa multi-unit complex. A multi-unit complex of the present invention caninclude, for example, at least 2, 3, 4, or 5 or more monomers. Themonomers and/or multi-unit complexes of the present invention may besoluble and may be isolated or purified to homogeneity. Monomer units ofa multi-unit complex may be different, homologous, substantiallyhomologous, or identical to one another. However, a multi-unit complexof the invention includes at least one monomer comprising a HRSpolypeptide as described herein or, in other embodiments, at least twoor more HRS polypeptides as described herein.

Covalently linked monomers can be linked directly (by bonds) orindirectly (e.g., via a linker). For directly linking the polypeptideherein, it may be beneficial to modify the polypeptides to enhancedimerization. For example, one or more amino acid residues of a HRSpolypeptide may be modified by the addition or substation by one or morecysteines. Methods for creating amino acid substitutions, such ascysteine substitutions, or other modifications to facilitate linking,are well known to those skilled in the art.

Certain embodiments of the present invention also contemplate the use ofmodified HRS polypeptides, including modifications that improve desiredcharacteristics of a HRS polypeptide, as described herein. Illustrativemodifications of HRS polypeptides of the invention include, but are notlimited to, chemical and/or enzymatic derivatizations at one or moreconstituent amino acids, including side chain modifications, backbonemodifications, and N- and/or C-terminal modifications includingacetylation, hydroxylation, methylation, amidation, and the attachmentof carbohydrate or lipid moieties, cofactors, and the like. Exemplarymodifications also include pegylation of a HRS polypeptide (see, e.g.,Veronese and Harris, Advanced Drug Delivery Reviews 54: 453-456, 2002,herein incorporated by reference).

In certain aspects, chemoselective ligation technology may be utilizedto modify HRS polypeptides of the invention, such as by attachingpolymers in a site-specific and controlled manner. Such technologytypically relies on the incorporation of chemoselective anchors into theprotein backbone by either chemical or recombinant means and subsequentmodification with a polymer carrying a complementary linker. As aresult, the assembly process and the covalent structure of the resultingprotein-polymer conjugate may be controlled, enabling the rationaloptimization of drug properties, such as efficacy and pharmacokineticproperties (see, e.g., Kochendoerfer, Current Opinion in ChemicalBiology 9:555-560, 2005).

The HRS polypeptides described herein may be prepared by any suitableprocedure known to those of skill in the art, such as by recombinanttechniques. For example, HRS polypeptides may be prepared by a procedureincluding the steps of: (a) preparing a construct comprising apolynucleotide sequence that encodes an HRS polypeptide and that isoperably linked to a regulatory element; (b) introducing the constructinto a host cell; (c) culturing the host cell to express the HRSpolypeptide; and (d) isolating the HRS polypeptide from the host cell.Recombinant HRS polypeptides can be conveniently prepared using standardprotocols as described for example in Sambrook, et al., (1989, supra),in particular Sections 16 and 17; Ausubel et al., (1994, supra), inparticular Chapters 10 and 16; and Coligan et al., Current Protocols inProtein Science (John Wiley & Sons, Inc. 1995-1997), in particularChapters 1, 5 and 6.

In addition to recombinant production methods, polypeptides of theinvention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc.85:2149-2154 (1963)). Protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be achieved, forexample, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the desiredmolecule.

Polynucleotide Compositions

The present invention also provides isolated polynucleotides that encodea HRS splice variant polypeptide of the invention, as described herein,as well as compositions comprising such polynucleotides. In certainembodiments, typically due to the singular nature of an HRS splicevariant, which combines exons in a new or exceptional way, the HRSpolynucleotides comprise a unique or exceptional splice junction.Exemplary reference HRS splice variant polynucleotides include SEQ IDNOS:5, 8, and 10, and variants and complements thereof.

Also included within the HRS polynucleotides of the present inventionare primers, probes, antisense oligonucleotides, and RNA interferenceagents that comprise all or a portion of these referencepolynucleotides, which are complementary to all or a portion of thesereference polynucleotides, or which specifically hybridize to thesereference polynucleotides, as described herein.

As used herein, the terms “DNA” and “polynucleotide” and “nucleic acid”refer to a DNA molecule that has been isolated free of total genomic DNAof a particular species. Therefore, a DNA segment encoding a polypeptiderefers to a DNA segment that contains one or more coding sequences yetis substantially isolated away from, or purified free from, totalgenomic DNA of the species from which the DNA segment is obtained.Included within the terms “DNA segment” and “polynucleotide” are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phagemids, phage,viruses, and the like.

As will be understood by those skilled in the art, the polynucleotidesequences of this invention can include genomic sequences, extra-genomicand plasmid-encoded sequences and smaller engineered gene segments thatexpress, or may be adapted to express, proteins, polypeptides, peptidesand the like. Such segments may be naturally isolated, or modifiedsynthetically by the hand of man.

As will be recognized by the skilled artisan, polynucleotides may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a HRS polypeptide of the invention or a portionthereof) or may comprise a variant, or a biological functionalequivalent of such a sequence. Polynucleotide variants may contain oneor more substitutions, additions, deletions and/or insertions, asfurther described below, preferably such that the desired non-canonicalactivity of the encoded polypeptide is not substantially diminishedrelative to the unmodified polypeptide. The effect on the activity ofthe encoded polypeptide may generally be assessed as described hereinand as would be recognized in the art.

The present invention further provides isolated polynucleotide fragmentscomprising various lengths of contiguous stretches of sequence identicalto or complementary to a HRS splice variant polynucleotide (e.g., SEQ IDNOs: 5, 8, or 10), as described herein, wherein the isolatedpolynucleotide encodes a HRS splice variant polypeptide of theinvention, or an active fragment or variant thereof.

For example, polynucleotides are provided by this invention that encodeat least about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 41, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, 160, 170 or more, contiguous amino acid residues of a HRSpolypeptide of the invention, as well as all intermediate lengths. Itwill be readily understood that “intermediate lengths”, in this context,means any length between the quoted values, such as 21, 22, 23, etc.;31, 32, 33, etc.; 41, 42, 43, etc.

The polynucleotides of the present invention, regardless of the lengthof the coding sequence itself, may be combined with other DNA sequences,such as promoters, polyadenylation signals, additional restrictionenzyme sites, multiple cloning sites, other coding segments, and thelike, such that their overall length may vary considerably. It istherefore contemplated that a polynucleotide fragment of almost anylength may be employed; with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol.

As noted above, certain embodiments relate to HRS polynucleotides thatencode an HRS polypeptide. Among other uses, these embodiments may beutilized to recombinantly produce a desired HRS polypeptide or variantthereof, or to express the HRS polypeptide in a selected cell orsubject. It will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that can encode a given HRS polypeptide asdescribed herein. Some of these polynucleotides may bear only limitedhomology to the reference nucleotide sequence. Nevertheless, suchpolynucleotides (i.e., degenerate variant polynucleotides) would beunderstood to encode the very same polypeptide. Accordingly,polynucleotides that vary due to differences in codon usage arespecifically contemplated by the present invention, for examplepolynucleotides that are optimized for human and/or primate codonselection.

Further, alleles of the genes comprising the polynucleotide sequencesprovided herein are within the scope of the present invention. Allelesare endogenous genes that are altered as a result of one or moremutations, such as deletions, additions and/or substitutions ofnucleotides. The resulting mRNA and protein may, but need not, have analtered structure or function. Alleles may be identified using standardtechniques (such as hybridization, amplification and/or databasesequence comparison).

Polynucleotides and fusions thereof may be prepared, manipulated and/orexpressed using any of a variety of well established techniques knownand available in the art. For example, polynucleotide sequences whichencode polypeptides of the invention, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of a HRS polypeptide of the invention in appropriate hostcells. Due to the inherent degeneracy of the genetic code, other DNAsequences that encode substantially the same or a functionallyequivalent amino acid sequence may be produced and these sequences maybe used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

Moreover, the polynucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterpolypeptide encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing,expression and/or activity of the gene product.

In order to express a desired polypeptide, a nucleotide sequenceencoding the polypeptide, or a functional equivalent, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook et al.,Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al.,Current Protocols in Molecular Biology (1989).

A variety of expression vector/host systems are known and may beutilized to contain and express polynucleotide sequences. These include,but are not limited to, microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transformed with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterialexpression vectors (e.g., Ti or pBR322 plasmids); or animal cellsystems, including viral-based expression systems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Theefficiency of expression may be enhanced by the inclusion of enhancerswhich are appropriate for the particular cell system which is used, suchas those described in the literature (Scharf. et al., Results Probl.Cell Differ. 20:125-162 (1994)).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such post-translationalmodifications of the polypeptide include, but are not limited to,acetylation, carboxylation, glycosylation, phosphorylation, lipidation,and acylation. Post-translational processing which cleaves a “prepro”form of the protein may also be used to facilitate correct insertion,folding and/or function. Different host cells such as CHO, HeLa, MDCK,HEK293, and W138, which have specific cellular machinery andcharacteristic mechanisms for such post-translational activities, may bechosen to ensure the correct modification and processing of the foreignprotein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a polynucleotide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler et al., Cell 11:223-232 (1977)) and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817-823 (1990)) geneswhich can be employed in tk- or aprt-cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler et al., Proc. Natl. Acad. Sci. U.S.A. 77:3567-70(1980)); npt, which confers resistance to the aminoglycosides, neomycinand G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); andals or pat, which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra).

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). These and otherassays are described, among other places, in Hampton et al., SerologicalMethods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med.158:1211-1216 (1983).

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides of theinvention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins.

In addition to recombinant production methods, polypeptides of theinvention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc.85:2149-2154 (1963)). Protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be achieved, forexample, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

According to another aspect of the invention, polynucleotides encodingpolypeptides of the invention may be delivered to a subject in vivo,e.g., using gene therapy techniques. Gene therapy refers generally tothe transfer of heterologous nucleic acids to the certain cells, targetcells, of a mammal, particularly a human, with a disorder or conditionsfor which such therapy is sought. The nucleic acid is introduced intothe selected target cells in a manner such that the heterologous DNA isexpressed and a therapeutic product encoded thereby is produced.

Various viral vectors that can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, adeno-associatedvirus (AAV), or, preferably, an RNA virus such as a retrovirus.Preferably, the retroviral vector is a derivative of a murine or avianretrovirus, or is a lentiviral vector. The preferred retroviral vectoris a lentiviral vector. Examples of retroviral vectors in which a singleforeign gene can be inserted include, but are not limited to: Moloneymurine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus(RSV). A number of additional retroviral vectors can incorporatemultiple genes. All of these vectors can transfer or incorporate a genefor a selectable marker so that transduced cells can be identified andgenerated. By inserting a zinc finger derived-DNA binding polypeptidesequence of interest into the viral vector, along with another gene thatencodes the ligand for a receptor on a specific target cell, forexample, the vector may be made target specific. Retroviral vectors canbe made target specific by inserting, for example, a polynucleotideencoding a protein (dimer). Illustrative targeting may be accomplishedby using an antibody to target the retroviral vector. Those of skill inthe art will know of, or can readily ascertain without undueexperimentation, specific polynucleotide sequences which can be insertedinto the retroviral genome to allow target specific delivery of theretroviral vector containing the zinc finger-nucleotide binding proteinpolynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsulation. Helper cell lines which havedeletions of the packaging signal include but are not limited to .PSI.2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced. The vector virions produced by thismethod can then be used to infect a tissue cell line, such as NIH 3T3cells, to produce large quantities of chimeric retroviral virions.

“Non-viral” delivery techniques for gene therapy can also be usedincluding, for example, DNA-ligand complexes, adenovirus-ligand-DNAcomplexes, direct injection of DNA, CaPO₄ precipitation, gene guntechniques, electroporation, liposomes, lipofection, and the like. Anyof these methods are widely available to one skilled in the art andwould be suitable for use in the present invention. Other suitablemethods are available to one skilled in the art, and it is to beunderstood that the present invention can be accomplished using any ofthe available methods of transfection. Lipofection can be accomplishedby encapsulating an isolated DNA molecule within a liposomal particleand contacting the liposomal particle with the cell membrane of thetarget cell. Liposomes are self-assembling, colloidal particles in whicha lipid bilayer, composed of amphiphilic molecules such as phosphatidylserine or phosphatidyl choline, encapsulates a portion of thesurrounding media such that the lipid bilayer surrounds a hydrophilicinterior. Unilammellar or multilammellar liposomes can be constructedsuch that the interior contains a desired chemical, drug, or, as in theinstant invention, an isolated DNA molecule.

Certain embodiments include polynucleotides that hybridize to areference HRS polynucleotide sequence, or to their complements, understringency conditions described below. As used herein, the term“hybridizes under low stringency, medium stringency, high stringency, orvery high stringency conditions” describes conditions for hybridizationand washing. Guidance for performing hybridization reactions can befound in Ausubel et al., (1998, supra), Sections 6.3.1-6.3.6. Aqueousand non-aqueous methods are described in that reference and either canbe used.

Reference herein to low stringency conditions include and encompass fromat least about 1% v/v to at least about 15% v/v formamide and from atleast about 1 M to at least about 2 M salt for hybridization at 42° C.,and at least about 1 M to at least about 2 M salt for washing at 42° C.Low stringency conditions also may include 1% Bovine Serum Albumin(BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65°C., and (i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄(pH 7.2), 5% SDS for washing at room temperature. One embodiment of lowstringency conditions includes hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by two washes in0.2×SSC, 0.1° A SDS at least at 50° C. (the temperature of the washescan be increased to 55° C. for low stringency conditions).

Medium stringency conditions include and encompass from at least about16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9 M salt for hybridization at 42° C., and at leastabout 0.1 M to at least about 0.2 M salt for washing at 55° C. Mediumstringency conditions also may include 1% Bovine Serum Albumin (BSA), 1mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C., and(i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2),5% SDS for washing at 60-65° C. One embodiment of medium stringencyconditions includes hybridizing in 6×SSC at about 45° C., followed byone or more washes in 0.2×SSC, 0.1% SDS at 60° C. High stringencyconditions include and encompass from at least about 31° A v/v to atleast about 50% v/v formamide and from about 0.01 M to about 0.15 M saltfor hybridization at 42° C., and about 0.01 M to about 0.02 M salt forwashing at 55° C.

High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 MNaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C., and (i) 0.2×SSC,0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS forwashing at a temperature in excess of 65° C. One embodiment of highstringency conditions includes hybridizing in 6×SSC at about 45° C.,followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C. Oneembodiment of very high stringency conditions includes hybridizing in0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washesin 0.2×SSC, 1% SDS at 65° C.

Other stringency conditions are well known in the art and a skilledartisan will recognize that various factors can be manipulated tooptimize the specificity of the hybridization. Optimization of thestringency of the final washes can serve to ensure a high degree ofhybridization. For detailed examples, see Ausubel et al., supra at pages2.10.1 to 2.10.16 and Sambrook et al. (1989, supra) at sections 1.101 to1.104.

While stringent washes are typically carried out at temperatures fromabout 42° C. to 68° C., one skilled in the art will appreciate thatother temperatures may be suitable for stringent conditions. Maximumhybridization rate typically occurs at about 20° C. to 25° C. below theT_(m) for formation of a DNA-DNA hybrid. It is well known in the artthat the T_(m) is the melting temperature, or temperature at which twocomplementary polynucleotide sequences dissociate. Methods forestimating T_(m) are well known in the art (see Ausubel et al., supra atpage 2.10.8).

In general, the T_(m) of a perfectly matched duplex of DNA may bepredicted as an approximation by the formula: T_(m)=81.5+16.6 (log₁₀M)+0.41 (% G+C)−0.63 (% formamide)−(600/length) wherein: M is theconcentration of Na⁺, preferably in the range of 0.01 molar to 0.4molar; % G+C is the sum of guanosine and cytosine bases as a percentageof the total number of bases, within the range between 30% and 75% G+C;% formamide is the percent formamide concentration by volume; length isthe number of base pairs in the DNA duplex. The T_(m) of a duplex DNAdecreases by approximately 1° C. with every increase of 1% in the numberof randomly mismatched base pairs. Washing is generally carried out atT_(m)−15° C. for high stringency, or T_(m)−30° C. for moderatestringency.

In one example of a hybridization procedure, a membrane (e.g., anitrocellulose membrane or a nylon membrane) containing immobilized DNAis hybridized overnight at 42° C. in a hybridization buffer (50%deionized formamide, 5×SSC, 5×Denhardt's solution (0.1% ficoll, 0.1%polyvinylpyrollidone and 0.1% bovine serum albumin), 0.1% SDS and 200mg/mL denatured salmon sperm DNA) containing a labeled probe. Themembrane is then subjected to two sequential medium stringency washes(i.e., 2×SSC, 0.1% SDS for 15 min at 45° C., followed by 2×SSC, 0.1% SDSfor 15 min at 50° C.), followed by two sequential higher stringencywashes (i.e., 0.2×SSC, 0.1% SDS for 12 min at 55° C. followed by 0.2×SSCand 0.1% SDS solution for 12 min at 65-68° C.

Embodiments of the present invention also include oligonucleotides,whether for detection, amplification, antisense therapies, or otherpurpose. For these and related purposes, the term “oligonucleotide” or“oligo” or “oligomer” is intended to encompass a singular“oligonucleotide” as well as plural “oligonucleotides,” and refers toany polymer of two or more of nucleotides, nucleosides, nucleobases orrelated compounds used as a reagent in the amplification methods of thepresent invention, as well as subsequent detection methods. Theoligonucleotide may be DNA and/or RNA and/or analogs thereof.

The term oligonucleotide does not necessarily denote any particularfunction to the reagent, rather, it is used generically to cover allsuch reagents described herein. An oligonucleotide may serve variousdifferent functions, e.g., it may function as a primer if it is capableof hybridizing to a complementary strand and can further be extended inthe presence of a nucleic acid polymerase, it may provide a promoter ifit contains a sequence recognized by an RNA polymerase and allows fortranscription, and it may function to prevent hybridization or impedeprimer extension if appropriately situated and/or modified. Anoligonucleotide may also function as a probe, or an antisense agent. Anoligonucleotide can be virtually any length, limited only by itsspecific function, e.g., in an amplification reaction, in detecting anamplification product of the amplification reaction, or in an antisenseor RNA interference application. Any of the oligonucleotides describedherein can be used as a primer, a probe, an antisense oligomer, or anRNA interference agent.

The term “primer” as used herein refers to a single-strandedoligonucleotide capable of acting as a point of initiation fortemplate-directed DNA synthesis under suitable conditions defined, forexample, by buffer and temperature, in the presence of four differentnucleoside triphosphates and an agent for polymerization, such as a DNAor RNA polymerase or reverse transcriptase. The length of the primer, inany given case, depends on, for example, the intended use of the primer,and generally ranges from about 15 to 30 nucleotides, although shorterand longer primers may be used. Short primer molecules generally requirecooler temperatures to form sufficiently stable hybrid complexes withthe template. A primer need not reflect the exact sequence of thetemplate but must be sufficiently complementary to hybridize with suchtemplate. The primer site is the area of the template to which a primerhybridizes. The primer pair is a set of primers including a 5′ upstreamprimer that hybridizes with the 5′ end of the sequence to be amplifiedand a 3′ downstream primer that hybridizes with the complement of the 3′end of the sequence to be amplified.

The term “probe” as used herein refers to a surface-immobilized moleculethat can be recognized by a particular target. See, e.g., U.S. Pat. No.6,582,908 for an example of arrays having all possible combinations ofprobes with 10, 12, and more bases. Probes and primers as used hereintypically comprise at least 10-15 contiguous nucleotides of a knownsequence. In order to enhance specificity, longer probes and primers mayalso be employed, such as probes and primers that comprise at least 20,25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 nucleotides of anHRS reference sequence or its complement. Probes and primers may beconsiderably longer than these examples, and it is understood that anylength supported by the knowledge in the art and the specification,including the tables, figures, and Sequence Listing, may be used.

Methods for preparing and using probes and primers are described in thereferences, for example Sambrook, J. et al. (1989) Molecular Cloning: ALaboratory Manual, 2.sup.nd ed., vol. 1-3, Cold Spring Harbor Press,Plainview N.Y.; Ausubel, F. M. et al. (1987) Current Protocols inMolecular Biology, Greene Publ. Assoc. & Wiley-Intersciences, New YorkN.Y.; Innis, M. et al. (1990) PCR Protocols. A Guide to Methods andApplications, Academic Press, San Diego Calif. PCR primer pairs can bederived from a known sequence, for example, by using computer programsintended for that purpose such as Primer (Version 0.5, 1991, WhiteheadInstitute for Biomedical Research, Cambridge Mass.).

Oligonucleotides for use as primers or probes may be selected usingsoftware known in the art. For example, OLIGO 4.06 software is usefulfor the selection of PCR primer pairs of up to 100 nucleotides each, andfor the analysis of oligonucleotides and larger polynucleotides of up to5,000 nucleotides from an input polynucleotide sequence of up to 32kilobases. The Primer3 primer selection program (available to the publicfrom the Whitehead Institute/MIT Center for Genome Research, CambridgeMass.) allows the user to input a “mispriming library,” in whichsequences to avoid as primer binding sites are user-specified. Theoligonucleotides and polynucleotide fragments identified by any of theabove selection methods are useful in hybridization technologies, forexample, as PCR or sequencing primers, microarray elements, or specificprobes to identify fully or partially complementary polynucleotides in asample of nucleic acids. Methods of oligonucleotide selection are notlimited to those described herein.

The terms “antisense oligomer” or “antisense compound” or “antisenseoligonucleotide” are used interchangeably and refer to a sequence ofcyclic subunits, each bearing a base-pairing moiety, linked byintersubunit linkages that allow the base-pairing moieties to hybridizeto a target sequence in a nucleic acid (typically an RNA) byWatson-Crick base pairing, to form a nucleic acid:oligomer heteroduplexwithin the target sequence, and typically thereby prevent translation ofthat RNA. Also included are methods of use thereof to modulateexpression of a selected HRS transcript, such as a splice variant orproteolytic fragment, and/or its corresponding polypeptide.

Antisense oligonucleotides may contain between about 8 and 40 subunits,typically about 8-25 subunits, and preferably about 12 to 25 subunits.In certain embodiments, oligonucleotides may have exact sequencecomplementarity to the target sequence or near complementarity, asdefined below. In certain embodiments, the degree of complementaritybetween the target and antisense targeting sequence is sufficient toform a stable duplex. The region of complementarity of the antisenseoligomers with the target RNA sequence may be as short as 8-11 bases,but is preferably 12-15 bases or more, e.g., 12-20 bases, or 12-25bases, including all integers in between these ranges. An antisenseoligomer of about 14-15 bases is generally long enough to have a uniquecomplementary sequence in targeting the selected HRS transcript.

In certain embodiments, antisense oligomers as long as 40 bases may besuitable, where at least a minimum number of bases, e.g., 10-12 bases,are complementary to the target sequence. In general, however,facilitated or active uptake in cells is optimized at oligomer lengthsless than about 30. For certain oligomers, described further below, anoptimum balance of binding stability and uptake generally occurs atlengths of 18-25 bases. Included are antisense oligomers (e.g., PNAs,LNAs, 2′-OMe, MOE, morpholinos) that consist of about 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, or 40 bases, in which at least about 6,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguousor non-contiguous bases are complementary to their HRS target sequence,or variants thereof.

In certain embodiments, antisense oligomers may be 100% complementary tothe HRS nucleic acid target sequence, or it may include mismatches,e.g., to accommodate variants, as long as a heteroduplex formed betweenthe oligomer and HRS nucleic acid target sequence is sufficiently stableto withstand the action of cellular nucleases and other modes ofdegradation which may occur in vivo. Oligomer backbones which are lesssusceptible to cleavage by nucleases are discussed below. Mismatches, ifpresent, are less destabilizing toward the end regions of the hybridduplex than in the middle. The number of mismatches allowed will dependon the length of the oligomer, the percentage of G:C base pairs in theduplex, and the position of the mismatch(es) in the duplex, according towell understood principles of duplex stability. Although such anantisense oligomer is not necessarily 100% complementary to the HRSnucleic acid target sequence, it is effective to stably and specificallybind to the target sequence, such that a biological activity of thenucleic acid target, e.g., expression of HRS protein(s), is modulated.

The stability of the duplex formed between an oligomer and a targetsequence is a function of the binding Tm and the susceptibility of theduplex to cellular enzymatic cleavage. The Tm of an antisenseoligonucleotide with respect to complementary-sequence RNA may bemeasured by conventional methods, such as those described by Hames etal., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108 or asdescribed in Miyada C. G. and Wallace R. B., 1987, Oligonucleotidehybridization techniques, Methods Enzymol. Vol. 154 pp. 94-107. Incertain embodiments, antisense oligomer may have a binding Tm, withrespect to a complementary-sequence RNA, of greater than bodytemperature and preferably greater than 50° C. Tm's in the range 60-80°C. or greater are preferred. According to well known principles, the Tmof an oligomer compound, with respect to a complementary-based RNAhybrid, can be increased by increasing the ratio of C:G paired bases inthe duplex, and/or by increasing the length (in base pairs) of theheteroduplex.

Antisense oligomers can be designed to block or inhibit translation ofmRNA or to inhibit natural pre-mRNA splice processing, or inducedegradation of targeted mRNAs, and may be said to be “directed to” or“targeted against” a target sequence with which it hybridizes. Incertain embodiments, the target sequence may include any coding ornon-coding sequence of an HRS mRNA transcript, and may thus by within anexon or within an intron. In certain embodiments, the target sequence isrelatively unique or exceptional among HRSs and is selective forreducing expression of a selected HRS proteolytic fragment or splicevariant. In certain embodiments, the target site includes a 3′ or 5′splice site of a pre-processed mRNA, or a branch point. The targetsequence for a splice site may include an mRNA sequence having its 5′end 1 to about 25 to about 50 base pairs downstream of a splice acceptorjunction or upstream of a splice donor junction in a preprocessed mRNA.In certain embodiments, a target sequence may include a splice junctionof an alternatively splice HRS mRNA, such as a splice junction that doesnot occur in the full-length HRS, or is unique or exceptional to thattranscript, in that it either does not occur or only seldom occurs inother HRS splice variants. An oligomer is more generally said to be“targeted against” a biologically relevant target, such as reference HRSpolynucleotide, when it is targeted against the nucleic acid of thetarget in the manner described herein.

A “subunit” of an oligonucleotide refers to one nucleotide (ornucleotide analog) unit. The term may refer to the nucleotide unit withor without the attached intersubunit linkage, although, when referringto a “charged subunit”, the charge typically resides within theintersubunit linkage (e.g., a phosphate or phosphorothioate linkage or acationic linkage).

The cyclic subunits of an oligonucleotide may be based on ribose oranother pentose sugar or, in certain embodiments, alternate or modifiedgroups. Examples of modified oligonucleotide backbones include, withoutlimitation, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Also contemplated are peptide nucleic acids(PNAs), locked nucleic acids (LNAs), 2′-O-Methyl oligonucleotides(2′-OMe), 2′-methoxyethoxy oligonucleotides (MOE), morpholinos, amongother oligonucleotides known in the art.

The purine or pyrimidine base pairing moiety is typically adenine,cytosine, guanine, uracil, thymine or inosine. Also included are basessuch as pyridin-4-one, pyridin-2-one, phenyl, pseudouracil,2,4,6-trime115thoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl,aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines(e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne,quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine,4-acetyltidine, 5-(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, β-D-galactosylqueosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguanosine,N6-methyladenosine, 7-methylguanosine,5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,5-methylcarbonyhnethyluridine, 5-methyloxyuridine,5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,β-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35,14090; Uhlman & Peyman, supra). By “modified bases” in this aspect ismeant nucleotide bases other than adenine (A), guanine (G), cytosine(C), thymine (T), and uracil (U), as illustrated above; such bases canbe used at any position in the antisense molecule. Persons skilled inthe art will appreciate that depending on the uses of the oligomers, Tsand Us are interchangeable. For instance, with other antisensechemistries such as 2′-O-methyl antisense oligonucleotides that are moreRNA-like, the T bases may be shown as U.

An oligonucleotide is typically complementary to a target sequence, suchas a target DNA or RNA. The terms “complementary” and “complementarity”refer to polynucleotides (i.e., a sequence of nucleotides) related bythe base-pairing rules. For example, the sequence “A-G-T,” iscomplementary to the sequence “T-C-A.” Complementarity may be “partial,”in which only some of the nucleic acids' bases are matched according tothe base pairing rules. Or, there may be “complete” or “total”complementarity (100%) between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. While perfect complementarity is often desired, someembodiments can include one or more but preferably 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatches withrespect to the target sequence. Variations at any location within theoligomer are included. In certain embodiments, variations in sequencenear the termini of an oligomer are generally preferable to variationsin the interior, and if present are typically within about 10, 9, 8, 7,6, 5, 4, 3, 2, or 1 nucleotides of the 5′ and/or 3′ terminus.

The term “target sequence” refers to a portion of the target RNA againstwhich the oligonucleotide is directed, that is, the sequence to whichthe oligonucleotide will hybridize by Watson-Crick base pairing of acomplementary sequence. In certain embodiments, the target sequence maybe a contiguous region of an HRS mRNA (e.g., a unique splice junction ofan HRS mRNA), or may be composed of non-contiguous regions of the mRNA.

The term “targeting sequence” or in certain embodiments “antisensetargeting sequence” refers to the sequence in an oligonucleotide that iscomplementary (meaning, in addition, substantially complementary) to thetarget sequence in the DNA or RNA target molecule. The entire sequence,or only a portion, of the antisense compound may be complementary to thetarget sequence. For example, in an oligonucleotide having 20-30 bases,about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, or 29 may be targeting sequences that arecomplementary to the target region. Typically, the targeting sequence isformed of contiguous bases, but may alternatively be formed ofnon-contiguous sequences that when placed together, e.g., from oppositeends of the oligonucleotide, constitute sequence that spans the targetsequence.

Target and targeting sequences are described as “complementary” to oneanother when hybridization occurs in an antiparallel configuration. Atargeting sequence may have “near” or “substantial” complementarity tothe target sequence and still function for the purpose of the presentinvention, that is, it may still be functionally “complementary.”

An oligonucleotide “specifically hybridizes” to a target polynucleotideif the oligomer hybridizes to a target (e.g., an HRS referencepolynucleotide or its complement) under physiological conditions, with aTm substantially greater than 45° C., preferably at least 50° C., andtypically 60° C.-80° C. or higher. Such hybridization preferablycorresponds to stringent hybridization conditions. At a given ionicstrength and pH, the Tm is the temperature at which 50% of a targetsequence hybridizes to a complementary polynucleotide. Again, suchhybridization may occur with “near” or “substantial” complementarity ofthe antisense oligomer to the target sequence, as well as with exactcomplementarity.

The terms specifically binds or specifically hybridizes refer generallyto an oligonucleotide probe or polynucleotide sequence that not onlybinds to its intended target gene sequence in a sample under selectedhybridization conditions, but does not bind significantly to othertarget sequences in the sample, and thereby discriminates between itsintended target and all other targets in the target pool. A probe thatspecifically hybridizes to its intended target sequence may also detectconcentration differences under the selected hybridization conditions,as described herein.

As noted above, certain oligonucleotides provided herein include peptidenucleic acids (PNAs). Also included are “locked nucleic acid” subunits(LNAs). The structures of LNAs are known in the art: for example,Wengel, et al., Chemical Communications (1998) 455; Tetrahedron (1998)54, 3607, and Accounts of Chem. Research (1999) 32, 301); Obika, et al.,Tetrahedron Letters (1997) 38, 8735; (1998) 39, 5401, and BioorganicMedicinal Chemistry (2008)16, 9230. Certain oligonucleotides maycomprise morpholino-based subunits bearing base-pairing moieties, joinedby uncharged or substantially uncharged linkages. The terms “morpholinooligomer” or “PMO” (phosphoramidate- or phosphorodiamidate morpholinooligomer) refer to an oligonucleotide analog composed of morpholinosubunit structures, where (i) the structures are linked together byphosphorus-containing linkages, one to three atoms long, preferably twoatoms long, and preferably uncharged or cationic, joining the morpholinonitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit,and (ii) each morpholino ring bears a purine or pyrimidine or anequivalent base-pairing moiety effective to bind, by base specifichydrogen bonding, to a base in a polynucleotide.

In certain embodiments, oligonucleotides can be prepared by stepwisesolid-phase synthesis, employing methods detailed in the referencescited above, and below with respect to the synthesis of oligonucleotideshaving a mixture or uncharged and cationic backbone linkages. In somecases, it may be desirable to add additional chemical moieties to theoligonucleotide, e.g., to enhance pharmacokinetics or to facilitatecapture or detection of the compound. Such a moiety may be covalentlyattached, typically to a terminus of the oligomer, according to standardsynthetic methods. For example, addition of a polyethylene glycol moietyor other hydrophilic polymer, e.g., one having 10-100 monomericsubunits, may be useful in enhancing solubility. One or more chargedgroups, e.g., anionic charged groups such as an organic acid, mayenhance cell uptake.

A variety of detectable molecules may be used to render anoligonucleotide detectable, such as a radioisotopes, fluorochromes,dyes, enzymes, nanoparticles, chemiluminescent markers, biotin, or othermonomer known in the art that can be detected directly (e.g., by lightemission) or indirectly (e.g., by binding of a fluorescently-labeledantibody).

Certain embodiments relate to RNA interference (RNAi) agents that targetone or more mRNA transcripts of an HRS reference polynucleotide,including fragments and variants thereof. Also included are methods ofuse thereof to modulate the levels of a selected HRS transcript, such asan HRS splice variant or proteolytic fragment.

The term “double-stranded” means two separate nucleic acid strandscomprising a region in which at least a portion of the strands aresufficiently complementary to hydrogen bond and form a duplex structure.The term “duplex” or “duplex structure” refers to the region of a doublestranded molecule wherein the two separate strands are substantiallycomplementary, and thus hybridize to each other. “dsRNA” refers to aribonucleic acid molecule having a duplex structure comprising twocomplementary and anti-parallel nucleic acid strands (i.e., the senseand antisense strands). Not all nucleotides of a dsRNA must exhibitWatson-Crick base pairs; the two RNA strands may be substantiallycomplementary. The RNA strands may have the same or a different numberof nucleotides.

The strands of a dsRNA are sufficiently complementary to hybridize toform a duplex structure. In certain embodiments, the complementary RNAstrand may be less than 30 nucleotides, less than 25 nucleotides inlength, or even 19 to 24 nucleotides in length. In certain aspects, thecomplementary nucleotide sequence may be 20-23 nucleotides in length, or22 nucleotides in length.

In certain embodiments, at least one of the RNA strands comprises anucleotide overhang of 1 to 4 nucleotides in length. In otherembodiments, one or both of the strands are blunt-ended. In certainembodiments, the dsRNA may further comprise at least one chemicallymodified nucleotide.

Certain embodiments of the present invention may comprise microRNAs.Micro-RNAs represent a large group of small RNAs produced naturally inorganisms, some of which regulate the expression of target genes.Micro-RNAs are formed from an approximately 70 nucleotidesingle-stranded hairpin precursor transcript by Dicer. (V. Ambros et al.Current Biology 13:807, 2003).

Certain embodiments may also employ short-interfering RNAs (siRNA). Eachstrand of an siRNA agent can be equal to or less than 35, 30, 25, 24,23, 22, 21, 20, 19, 18, 17, 16, or 15 nucleotides in length. The strandis preferably at least 19 nucleotides in length. For example, eachstrand can be between 21 and 25 nucleotides in length. Preferred siRNAagents have a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25nucleotide pairs, and one or more overhangs, preferably one or two 3′overhangs, of 2-3 nucleotides.

A “single strand RNAi agent” as used herein, is an RNAi agent which ismade up of a single molecule. It may include a duplexed region, formedby intra-strand pairing, e.g., it may be, or include, a hairpin orpan-handle structure. A single strand RNAi agent is at least 14, andmore preferably at least 15, 20, 25, 29, 35, 40, or 50 nucleotides inlength. It is preferably less than 200, 100, or 60 nucleotides inlength.

Hairpin RNAi modulating agents may have a duplex region equal to or atleast 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplexregion may preferably be equal to or less than 200, 100, or 50, inlength. Certain ranges for the duplex region are 15-30, 17 to 23, 19 to23, and 19 to 21 nucleotides pairs in length. The hairpin may have asingle strand overhang or terminal unpaired region, preferably the 3′,and preferably of the antisense side of the hairpin. In certainembodiments, overhangs are 2-3 nucleotides in length.

The present invention further encompasses oligonucleotides employingribozymes. Also included are vector delivery systems that are capable ofexpressing the HRS-targeting sequences described herein. Included arevectors that express siRNA or other duplex-forming RNA interferencemolecules. Exemplary delivery systems may include viral vector systems(i.e., viral-mediated transduction) including, but not limited to,retroviral (e.g., lentiviral) vectors, adenoviral vectors,adeno-associated viral vectors, and herpes viral vectors, among othersknown in the art.

Oligonucleotides and RNAi agents that target one or more portions of anHRS polynucleotide reference sequence or its complement may be used inany of the therapeutic, diagnostic, or drug screening methods describedherein and apparent to persons skilled in the art.

Antibody Compositions, Fragments Thereof and Other Binding Agents

According to another aspect, the present invention further providesbinding agents, such as antibodies, antigen-binding fragments thereof,soluble receptors, aptamers, small molecules, etc., that exhibit bindingspecificity for a HRS splice variant polypeptide or its cellular bindingpartner as disclosed herein, or to a portion, variant or derivativethereof, and methods of using same.

In some embodiments, such binding agents will be effective formodulating one or more of the non-canonical activities mediated by a HRSpolypeptide of the invention. In certain other embodiments, for example,the binding agent is one that binds to a HRS polypeptide of theinvention and inhibits its ability to bind to one or more of itscellular binding partners. Accordingly, such binding agents may be usedto treat or prevent diseases, disorders or other conditions that aremediated by, or modulated by, a HRS polypeptide of the invention byantagonizing it activity. In certain embodiments, for example, thebinding agent binds to the cellular binding partner of an HRSpolypeptide, and mimics the HRS polypeptide activity, such as byincreasing or agonizing the non-canonical activity mediated by the HRSpolypeptide. Accordingly, such binding agents may be used to diagnose,treat, or prevent diseases, disorders or other conditions that aremediated by an HRS polypeptide of the invention, such as by antagonizingor agonizing its activity partially or fully.

An binding agent such as an antibody, or antigen-binding fragmentthereof, is said to “specifically bind,” “immunologically bind,” and/oris “immunologically reactive” to a polypeptide of the invention if itreacts at a detectable level (within, for example, an ELISA assay) withthe polypeptide, and does not react detectably with unrelatedpolypeptides under similar conditions.

Immunological binding, as used in this context, generally refers to thenon-covalent interactions of the type which occur between animmunoglobulin molecule and an antigen for which the immunoglobulin isspecific. The strength, or affinity of immunological bindinginteractions can be expressed in terms of the dissociation constant(K_(d)) of the interaction, wherein a smaller K_(d) represents a greateraffinity. Immunological binding properties of selected polypeptides canbe quantified using methods well known in the art. One such methodentails measuring the rates of antigen-binding site/antigen complexformation and dissociation, wherein those rates depend on theconcentrations of the complex partners, the affinity of the interaction,and on geometric parameters that equally influence the rate in bothdirections. Thus, both the “on rate constant” (K_(on)) and the “off rateconstant” (K_(off)) can be determined by calculation of theconcentrations and the actual rates of association and dissociation. Theratio of K_(off)/K_(on) enables cancellation of all parameters notrelated to affinity, and is thus equal to the dissociation constantK_(d). See, e.g., Davies et al. (1990) Annual Rev. Biochem. 59:439-473.

An “antigen-binding site,” or “binding portion” of an antibody, refersto the part of the immunoglobulin molecule that participates in antigenbinding. The antigen binding site is formed by amino acid residues ofthe N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”)chains. Three highly divergent stretches within the V regions of theheavy and light chains are referred to as “hypervariable regions” whichare interposed between more conserved flanking stretches known as“framework regions,” or “FRs”. Thus the term “FR” refers to amino acidsequences which are naturally found between and adjacent tohypervariable regions in immunoglobulins. In an antibody molecule, thethree hypervariable regions of a light chain and the three hypervariableregions of a heavy chain are disposed relative to each other in threedimensional space to form an antigen-binding surface. Theantigen-binding surface is complementary to the three-dimensionalsurface of a bound antigen, and the three hypervariable regions of eachof the heavy and light chains are referred to as“complementarity-determining regions,” or “CDRs.”

A binding agent may be, for example, a ribosome, with or without apeptide component, an RNA molecule or a polypeptide. In a preferredembodiment, a binding agent is an antibody or an antigen-bindingfragment thereof. Antibodies may be prepared by any of a variety oftechniques known to those of ordinary skill in the art. See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. Monoclonal antibodies specific for a polypeptide ofinterest may be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.The polypeptides of this invention may be used in the purificationprocess in, for example, an affinity chromatography step.

An “Fv” fragment can be produced by preferential proteolytic cleavage ofan IgM, and on rare occasions IgG or IgA immunoglobulin molecule. Fvfragments are, however, more commonly derived using recombinanttechniques known in the art. The Fv fragment includes a non-covalentV_(H)::V_(L) heterodimer including an antigen-binding site which retainsmuch of the antigen recognition and binding capabilities of the nativeantibody molecule. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich etal. (1980) Biochem 19:4091-4096.

A single chain Fv (“sFv”) polypeptide is a covalently linkedV_(H)::V_(L) heterodimer which is expressed from a gene fusion includingV_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker.Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. Anumber of methods have been described to discern chemical structures forconverting the naturally aggregated—but chemically separated—light andheavy polypeptide chains from an antibody V region into an sFv moleculewhich will fold into a three dimensional structure substantially similarto the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

Each of the above-described molecules includes a heavy chain and a lightchain CDR set, respectively interposed between a heavy chain and a lightchain FR set which provide support to the CDRS and define the spatialrelationship of the CDRs relative to each other. As used herein, theterm “CDR set” refers to the three hypervariable regions of a heavy orlight chain V region. Proceeding from the N-terminus of a heavy or lightchain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3”respectively. An antigen-binding site, therefore, includes six CDRs,comprising the CDR set from each of a heavy and a light chain V region.A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) isreferred to herein as a “molecular recognition unit.” Crystallographicanalysis of a number of antigen-antibody complexes has demonstrated thatthe amino acid residues of CDRs form extensive contact with boundantigen, wherein the most extensive antigen contact is with the heavychain CDR3. Thus, the molecular recognition units are primarilyresponsible for the specificity of an antigen-binding site.

As used herein, the term “FR set” refers to the four flanking amino acidsequences which frame the CDRs of a CDR set of a heavy or light chain Vregion. Some FR residues may contact bound antigen; however, FRs areprimarily responsible for folding the V region into the antigen-bindingsite, particularly the FR residues directly adjacent to the CDRS. WithinFRs, certain amino residues and certain structural features are veryhighly conserved. In this regard, all V region sequences contain aninternal disulfide loop of around 90 amino acid residues. When the Vregions fold into a binding-site, the CDRs are displayed as projectingloop motifs which form an antigen-binding surface. It is generallyrecognized that there are conserved structural regions of FRs whichinfluence the folded shape of the CDR loops into certain “canonical”structures—regardless of the precise CDR amino acid sequence. Further,certain FR residues are known to participate in non-covalent interdomaincontacts which stabilize the interaction of the antibody heavy and lightchains.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent V regions and theirassociated CDRs fused to human constant domains (Winter et al. (1991)Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA86:4220-4224; Shaw et al. (1987) J Immunol. 138:4534-4538; and Brown etal. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a humansupporting FR prior to fusion with an appropriate human antibodyconstant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyenet al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature321:522-525), and rodent CDRs supported by recombinantly veneered rodentFRs (European Patent Publication No. 519,596, published Dec. 23, 1992).These “humanized” molecules are designed to minimize unwantedimmunological response toward rodent antihuman antibody molecules whichlimits the duration and effectiveness of therapeutic applications ofthose moieties in human recipients.

As noted above, “peptides” are included as binding agents. The termpeptide typically refers to a polymer of amino acid residues and tovariants and synthetic analogues of the same. In certain embodiments,the term “peptide” refers to relatively short polypeptides, includingpeptides that consist of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids,including all integers and ranges (e.g., 5-10, 8-12, 10-15) in between,and interact with an HRS polypeptide, its cellular binding partner, orboth. Peptides can be composed of naturally-occurring amino acids and/ornon-naturally occurring amino acids, as described herein.

A binding agent may include a peptide mimetic or other small molecule. A“small molecule” refers to an organic compound that is of synthetic orbiological origin (biomolecule), but is typically not a polymer. Organiccompounds refer to a large class of chemical compounds whose moleculescontain carbon, typically excluding those that contain only carbonates,simple oxides of carbon, or cyanides. A “biomolecule” refers generallyto an organic molecule that is produced by a living organism, includinglarge polymeric molecules (biopolymers) such as peptides,polysaccharides, and nucleic acids as well, and small molecules such asprimary secondary metabolites, lipids, phospholipids, glycolipids,sterols, glycerolipids, vitamins, and hormones. A “polymer” refersgenerally to a large molecule or macromolecule composed of repeatingstructural units, which are typically connected by covalent chemicalbond.

In certain embodiments, a small molecule has a molecular weight of lessthan 1000 Daltons, typically between about 300 and 700 Daltons, andincluding about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,500, 650, 600, 750, 700, 850, 800, 950, or 1000 Daltons.

Aptamers are also included as binding agents. Examples of aptamersincluded nucleic acid aptamers (e.g., DNA aptamers, RNA aptamers) andpeptide aptamers. Nucleic acid aptamers refer generally to nucleic acidspecies that have been engineered through repeated rounds of in vitroselection or equivalent method, such as SELEX (systematic evolution ofligands by exponential enrichment), to bind to various molecular targetssuch as small molecules, proteins, nucleic acids, and even cells,tissues and organisms. Hence, included are nucleic acid aptamers thatbind to the HRS polypeptides described herein and/or their cellularbinding partners.

Peptide aptamers typically include a variable peptide loop attached atboth ends to a protein scaffold, a double structural constraint thattypically increases the binding affinity of the peptide aptamer tolevels comparable to that of an antibody's (e.g., in the nanomolarrange). In certain embodiments, the variable loop length may be composedof about 10-20 amino acids (including all integers in between), and thescaffold may include any protein that has good solubility and compacityproperties. Certain exemplary embodiments may utilize the bacterialprotein Thioredoxin-A as a scaffold protein, the variable loop beinginserted within the reducing active site (-Cys-Gly-Pro-Cys-loop in thewild protein), with the two cysteines lateral chains being able to forma disulfide bridge. Hence, included are peptide aptamers that bind tothe HRS polypeptides described herein and/or their cellular bindingpartners. Peptide aptamer selection can be performed using differentsystems known in the art, including the yeast two-hybrid system.

As noted above, the HRS polypeptides and binding agents of the presentinvention can be used in any of the diagnostic, drug discovery, ortherapeutic methods described herein.

In another embodiment of the invention, binding agents such asmonoclonal antibodies of the present invention may be coupled to one ormore agents of interest. For example, a therapeutic agent may be coupled(e.g., covalently bonded) to a suitable monoclonal antibody eitherdirectly or indirectly (e.g., via a linker group). A direct reactionbetween an agent and an antibody is possible when each possesses asubstituent capable of reacting with the other. For example, anucleophilic group, such as an amino or sulfhydryl group, on one may becapable of reacting with a carbonyl-containing group, such as ananhydride or an acid halide, or with an alkyl group containing a goodleaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958, to Rodwell et al.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule, or linkers that provide multiple sites for attachmentcan be used.

Formulation and Administration

The compositions of the invention (e.g., HRS splice variantpolypeptides, polynucleotides, binding agents, antibodies, etc.) aregenerally formulated in pharmaceutically-acceptable orphysiologically-acceptable solutions for administration to a cell,tissue or animal, either alone, or in combination with one or more othermodalities of therapy. It will also be understood that, if desired, thecompositions of the invention may be administered in combination withother agents as well, such as, e.g., other proteins or polypeptides orvarious pharmaceutically-active agents. There is virtually no limit toother components that may also be included in the compositions, providedthat the additional agents do not adversely affect the effects desiredto be achieved with a HRS composition of the invention.

In the pharmaceutical compositions of the invention, formulation ofpharmaceutically-acceptable excipients and carrier solutions iswell-known to those of skill in the art, as is the development ofsuitable dosing and treatment approaches for using the particularcompositions described herein in a variety of treatment regimens,including e.g., oral, parenteral, intravenous, intranasal, intracranialand intramuscular administration and formulation.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to a subject. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally as described,for example, in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (eachspecifically incorporated herein by reference in its entirety).Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form should be sterileand should be fluid to the extent that easy syringability exists. Itshould be stable under the conditions of manufacture and storage andshould be preserved against the contaminating action of microorganisms,such as bacteria and fungi. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be facilitated by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion (see, e.g., Remington's PharmaceuticalSciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

Sterile injectable solutions can be prepared by incorporating the activecompounds in the required amount in the appropriate solvent with thevarious other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug-release capsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human or other mammal. The preparationof an aqueous composition that contains a protein as an activeingredient is well understood in the art. Typically, such compositionsare prepared as injectables, either as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid prior toinjection can also be prepared. The preparation can also be emulsified.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, polynucleotides, and peptidecompositions directly to the lungs via nasal aerosol sprays has beendescribed e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (eachspecifically incorporated herein by reference in its entirety).Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S.Pat. No. 5,725,871, specifically incorporated herein by reference in itsentirety) are also well-known in the pharmaceutical arts. Likewise,transmucosal drug delivery in the form of a polytetrafluoroethylenesupport matrix is described in U.S. Pat. No. 5,780,045 (specificallyincorporated herein by reference in its entirety).

In certain embodiments, the delivery may occur by use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the compositions ofthe present invention may be formulated for delivery either encapsulatedin a lipid particle, a liposome, a vesicle, a nanosphere, a nanoparticleor the like. The formulation and use of such delivery vehicles can becarried out using known and conventional techniques.

Kits Comprising Compositions of the Invention

The invention, in other aspects, provides kits comprising one or morecontainers filled with one or more of the HRS splice variantpolypeptides, polynucleotides, antibodies, compositions thereof, etc.,of the invention, as described herein. The kits can include writteninstructions on how to use such compositions (e.g., to modulate cellularsignaling, angiogenesis, cancer, inflammatory conditions, etc.).

The kits herein may also include a one or more additional therapeuticagents or other components suitable or desired for the indication beingtreated. An additional therapeutic agent may be contained in a secondcontainer, if desired. Examples of additional therapeutic agentsinclude, but are not limited to antineoplastic agents, anti-inflammatoryagents, antibacterial agents, antiviral agents, angiogenic agents, etc.

The kits herein can also include one or more syringes or othercomponents necessary or desired to facilitate an intended mode ofdelivery (e.g., stents, implantable depots, etc.).

Methods of Use

Embodiments of the present invention also include methods of using theHRS compositions or “agents” described herein for diagnostic, drugdiscovery, and/or therapeutic purposes. The term HRS “agents” refersgenerally to the HRS polynucleotides, HRS polypeptides, binding agents,and other compounds described herein. For diagnostic purposes, the HRSagents can be used in a variety of non-limiting ways, such as todistinguish between different cell types or different cellular states,or to identify subjects having a relevant disease or condition. For drugdiscovery purposes, the HRS agents can be used to identify one or morecellular “binding partners” of an HRS polypeptide, characterize one ormore “non-canonical” activities of an HRS polypeptide, identify agentsthat selectively or non-selectively agonize or antagonize theinteraction of an HRS polypeptide with its binding partner(s), and/oridentify agents that selectively or non-selectively agonize orantagonize one or more “non-canonical” activities of an HRS polypeptide.For therapeutic purposes, the HRS agents or compositions provided hereincan be used to treat a variety of diseases or conditions, detailedbelow.

A. Diagnostics

As noted above, HRS agents described herein can be used in diagnosticassays. These embodiments include the detection of the HRSpolynucleotide sequence(s) or corresponding polypeptide sequence(s) orportions thereof of one or more newly identified HRS splice variants,and/or one or more splice junctions of those splice variants. In certainembodiments, the polynucleotide or corresponding polypeptide sequence(s)of at least one of the splice junctions is unique to that particular HRSsplice variant. In certain embodiments, the presence or levels of one ormore newly identified HRS splice variants, as typically characterized bythe polynucleotide or corresponding polypeptide sequence of their splicejunctions, associate or correlate with one or more cellular types orcellular states. Hence, as noted above, the presence or levels of an HRSsplice variant or its splice junction can be used to distinguish betweendifferent cellular types or different cellular states. The presence orlevels of HRS splice variants or their splice junctions can be detectedaccording to polynucleotide and/or polypeptide-based diagnostictechniques.

Certain of the methods provided herein rely on the differentialexpression of an HRS splice variant to characterize the condition orstate of a cell, tissue, or subject, and to distinguish it from anothercell, tissue, or subject. Non-limiting examples include methods ofdetecting the presence or levels of an HRS splice variant or its splicejunction in a biological sample to distinguish between cells or tissuesof different species, cells of different tissues or organs, cellulardevelopmental states such as neonatal and adult, cellulardifferentiation states, conditions such as healthy, diseased andtreated, intracellular and extracellular fractions, in addition toprimary cell cultures and other cell cultures, such as immortalized cellcultures.

Differential expression refers generally to a statistically significantdifference in one or more gene expression levels of an HRSpolynucleotide or polypeptide sequence compared to the expression levelsof the same sequence in an appropriate control. The statisticallysignificant difference may relate to either an increase or a decrease inexpression levels, as measured by RNA levels, protein levels, proteinfunction, or any other relevant measure of gene expression such as thosedescribed herein.

A result is typically referred to as statistically significant if it isunlikely to have occurred by chance. The significance level of a test orresult relates traditionally to a frequentist statistical hypothesistesting concept. In simple cases, statistical significance may bedefined as the probability of making a decision to reject the nullhypothesis when the null hypothesis is actually true (a decision knownas a Type I error, or “false positive determination”). This decision isoften made using the p-value: if the p-value is less than thesignificance level, then the null hypothesis is rejected. The smallerthe p-value, the more significant the result. Bayes factors may also beutilized to determine statistical significance (see, e.g., Goodman S.,Ann Intern Med 130:1005-13, 1999).

In more complicated, but practically important cases, the significancelevel of a test or result may reflect an analysis in which theprobability of making a decision to reject the null hypothesis when thenull hypothesis is actually true is no more than the stated probability.This type of analysis allows for those applications in which theprobability of deciding to reject may be much smaller than thesignificance level for some sets of assumptions encompassed within thenull hypothesis.

In certain exemplary embodiments, statistically significant differentialexpression may include situations wherein the expression level of agiven HRS sequence provides at least about a 1.2×, 1.3×, 1.4×, 1.5×,1.6×, 1.7×, 1.8×, 1.9×. 2.0×, 2.2×, 2.4×, 2.6×, 2.8×, 3.0×, 4.0×, 5.0×,6.0×, 7.0×, 8.0×, 9.0×, 10.0×, 15.0×, 20.0×, 50.0×, 100.0×, or greaterdifference in expression (i.e., differential expression that may behigher or lower expression) in a suspected biological sample as comparedto an appropriate control, including all integers and decimal points inbetween (e.g., 1.24×, 1.25×, 2.1×, 2.5×, 60.0×, 75.0×, etc.). In certainembodiments, statistically significant differential expression mayinclude situations wherein the expression level of a given HRS sequenceprovides at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000 percent (%) or greater difference inexpression (i.e., differential expression that may be higher or lower)in a suspected biological sample as compared to an appropriate control,including all integers and decimal points in between.

As an additional example, differential expression may also be determinedby performing Z-testing, i.e., calculating an absolute Z score, asdescribed herein and known in the art (see Example 1). Z-testing istypically utilized to identify significant differences between a samplemean and a population mean. For example, as compared to a standardnormal table (e.g., a control tissue), at a 95% confidence interval(i.e., at the 5% significance level), a Z-score with an absolute valuegreater than 1.96 indicates non-randomness. For a 99% confidenceinterval, if the absolute Z is greater than 2.58, it means that p<0.01,and the difference is even more significant—the null hypothesis can berejected with greater confidence. In these and related embodiments, anabsolute Z-score of 1.96, 2, 2.58, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or more, including all decimal points inbetween (e.g., 10.1, 10.6, 11.2, etc.), may provide a strong measure ofstatistical significance. In certain embodiments, an absolute Z-score ofgreater than 6 may provide exceptionally high statistical significance.

Substantial similarly relates generally to the lack of a statisticallysignificant difference in the expression levels between the biologicalsample and the reference control. Examples of substantially similarexpression levels may include situations wherein the expression level ofa given SSCIGS provides less than about a 0.05×, 0.1×, 0.2×, 0.3×, 0.4×,0.5×, 0.6×, 0.7×, 0.8×, 0.9×. 1.0×, 1.1×, 1.2×, 1.3×, or 1.4× differencein expression (i.e., differential expression that may be higher or lowerexpression) in a suspected biological sample as compared to a referencesample, including all decimal points in between (e.g., 0.15×, 0.25×,0.35×, etc.). In certain embodiments, differential expression mayinclude situations wherein the expression level of a given HRS sequenceprovides less than about 0.25. 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 percent (%) difference inexpression (i.e., differential expression that may be higher or lower)in a suspected biological sample as compared to a reference sample,including all decimal points in between.

In certain embodiments, such as when using an Affymetrix Microarray tomeasure the expression levels of an HRS polynucleotide or polypeptidesequence, differential expression may also be determined by the meanexpression value summarized by Affymetrix Microarray Suite 5 software(Affymetrix, Santa Clara, Calif.), or other similar software, typicallywith a scaled mean expression value of 1000.

Embodiments of the present invention include methods of detecting thepresence or levels of an HRS polynucleotide or polypeptide referencesequence or a portion thereof to distinguish between cells, tissues, orother biological samples of a different organism or species, wherein thepresence or levels of that sequence associates with a selected organismor species. General examples include methods of distinguishing betweenhumans and any combination of bacteria, fungi, plants, and othernon-human animals. Included within animals are methods of distinguishingbetween humans and any combination of vertebrates and invertebrates,including vertebrates such as fish, amphibians, reptiles, birds, andnon-human mammals, and invertebrates such as insects, mollusks,crustaceans, and corals. Included within non-human mammals are methodsof distinguishing between humans and any combination of non-humanmammals from the Order Afrosoricida, Macroscelidea, Tubulidentata,Hyracoidea, Proboscidea, Sirenia, Cingulata, Pilosa, Scandentia,Dermoptera, Primates, Rodentia, Lagomorpha, Erinaceomorpha,Soricomorpha, Chiroptera, Pholidota, Cetacea, Carnivora, Perissodactyla,or Artiodactyla. Included within the Primate Order are monkeys, apes,gorillas, and chimpanzees, among others known in the art. Accordingly,the presence or levels of an HRS polynucleotide or polypeptide referencesequence or variant, as described herein, may be used to identify thesource of a given biological sample, such as a cell, tissue, or organ,by distinguishing between any combination of these organisms, or bydistinguishing between humans and any one or more of these organisms,such as a panel of organisms. In certain embodiments, the source of agiven biological sample may also be determined by comparing the presenceor levels of an HRS sequence or a portion thereof to a pre-determinedvalue.

Embodiments of the present invention include methods of detecting thepresence or levels of an HRS polynucleotide or polypeptide referencesequence or a portion thereof to distinguish between cells or otherbiological samples that originate from different tissues or organs.Non-limiting examples include methods of distinguishing between a cellor other biological sample that originates from any combination of skin(e.g., dermis, epidermis, subcutaneous layer), hair follicles, nervoussystem (e.g., brain, spinal cord, peripheral nerves), auditory system orbalance organs (e.g., inner ear, middle ear, outer ear), respiratorysystem (e.g., nose, trachea, lungs), gastroesophogeal tissues, thegastrointestinal system (e.g., mouth, esophagus, stomach, smallintestines, large intestines, rectum), vascular system (e.g., heart,blood vessels and arteries), liver, gallbladder, lymphatic/immune system(e.g., lymph nodes, lymphoid follicles, spleen, thymus, bone marrow),uro-genital system (e.g., kidneys, ureter, bladder, urethra, cervix,Fallopian tubes, ovaries, uterus, vulva, prostate, bulbourethral glands,epidiymis, prostate, seminal vesicles, testicles), musculoskeletalsystem (e.g., skeletal muscles, smooth muscles, bone, cartilage,tendons, ligaments), adipose tissue, mammaries, and the endocrine system(e.g., hypothalamus, pituitary, thyroid, pancreas, adrenal glands).Hence, based on the association of an HRS polynucleotide or polypeptidesequence as described herein, these methods may be used to identify orcharacterize the tissue or organ from which a cell or other biologicalsample is derived.

Embodiments of the present invention include methods of detecting thepresence or levels of an HRS polynucleotide or polypeptide referencesequence or a portion thereof to distinguish between or characterize thedevelopmental or differentiation state of the cell. Also included aremethods of differentiating between germ cells, stem cells, and somaticcells. Examples of developmental states include neonatal and adult.Examples of cellular differentiation states include all of the discreetand identifiable stages between a totipotent cell, a pluripotent cell, amultipotent progenitor stem cell and a mature, fully differentiatedcell.

A totipotent cell has total potential, typically arises during sexualand asexual reproduction, and includes and spores and zygotes, though incertain instances cells can dedifferentiate and regain totipotency. Apluripotent cell includes a stem cell that has the potential todifferentiate into any of the three germ layers, including the endoderm(interior stomach lining, gastrointestinal tract, the lungs), themesoderm (muscle, bone, blood, urogenital), and the ectoderm (epidermaltissues and nervous system). Multipotent progenitor cells are typicallycapable of differentiating into a limited number of tissue types.Examples of multipotent cells include, without limitation, hematopoieticstem cells (adult stem cells) from the bone marrow that give rise toimmune cells such as red blood cells, white blood cells, and platelets,mesenchymal stem cells (adult stem cells) from the bone marrow that giverise to stromal cells, fat cells, and various types of bone cells,epithelial stem cells (progenitor cells) that give rise to the varioustypes of skin cells, and muscle satellite cells (progenitor cells) thatcontribute to differentiated muscle tissue. Accordingly, the presence orlevels of particular HRS polynucleotide or polypeptide sequence (e.g.,splice junction of an HRS splice variant), can be used to distinguishbetween or characterize the above-noted cellular differentiation states,as compared to a control or a predetermined level.

Embodiments of the present invention include methods of detecting thepresence or levels of an HRS polynucleotide or polypeptide referencesequence to characterize or diagnose the condition or a cell, tissue,organ, or subject, in which that condition may be characterized ashealthy, diseased, at risk for being diseased, or treated. For suchdiagnostic purposes, the term “diagnostic” or “diagnosed” includesidentifying the presence or nature of a pathologic condition,characterizing the risk of developing such a condition, and/or measuringthe change (or no change) of a pathologic condition in response totherapy. Diagnostic methods may differ in their sensitivity andspecificity. In certain embodiments, the “sensitivity” of a diagnosticassay refers to the percentage of diseased cells, tissues or subjectswhich test positive (percent of “true positives”). Diseased cells,tissues or subjects not detected by the assay are typically referred toas “false negatives.” Cells, tissues or subjects that are not diseasedand which test negative in the assay may be termed “true negatives.” Incertain embodiments, the “specificity” of a diagnostic assay may bedefined as one (1) minus the false positive rate, where the “falsepositive” rate is defined as the proportion of those samples or subjectswithout the disease and which test positive. While a particulardiagnostic method may not provide a definitive diagnosis of a condition,it suffices if the method provides a positive indication that aids indiagnosis.

In certain instances, the presence or risk of developing a pathologiccondition can be diagnosed by comparing the presence or levels of one ormore selected HRS polynucleotide or polypeptide reference sequences orportions thereof that correlate with the condition, whether by increasedor decreased levels, as compared to a suitable control. A “suitablecontrol” or “appropriate control” includes a value, level, feature,characteristic, or property determined in a cell or other biologicalsample of a tissue or organism, e.g., a control or normal cell, tissueor organism, exhibiting, for example, normal traits, such as the absenceof the condition. In certain embodiments, a “suitable control” or“appropriate control” is a predefined value, level, feature,characteristic, or property. Other suitable controls will be apparent topersons skilled in the art. Examples of diseases and conditions aredescribed elsewhere herein.

Embodiments of the present invention include HRS polynucleotide ornucleic acid-based detection techniques, which offer certain advantagesdue to sensitivity of detection. Hence, certain embodiments relate tothe use or detection of HRS polynucleotides as part of a diagnosticmethod or assay. The presence and/or levels of HRS polynucleotides maybe measured by any method known in the art, including hybridizationassays such as Northern blot, quantitative or qualitative polymerasechain reaction (PCR), quantitative or qualitative reverse transcriptasePCR (RT-PCR), microarray, dot or slot blots, or in situ hybridizationsuch as fluorescent in situ hybridization (FISH), among others. Certainof these methods are described in greater detail below.

HRS polynucleotides such as DNA and RNA can be collected and/orgenerated from blood, biological fluids, tissues, organs, cell lines, orother relevant sample using techniques known in the art, such as thosedescribed in Kingston. (2002 Current Protocols in Molecular Biology,Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, NY (see, e.g.,as described by Nelson et al. Proc Natl Acad Sci USA, 99: 11890-11895,2002) and elsewhere.

Complementary DNA (cDNA) libraries can be generated using techniquesknown in the art, such as those described in Ausubel et al. (2001Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & JohnWiley & Sons, Inc., NY, NY); Sambrook et al. (1989 Molecular Cloning,Second Ed., Cold Spring Harbor Laboratory, Plainview, N.Y.); Maniatis etal. (1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview,N.Y.) and elsewhere.

Certain embodiments may employ hybridization methods for detecting HRSpolynucleotide sequences. Methods for conducting polynucleotidehybridization assays have been well developed in the art. Hybridizationassay procedures and conditions will vary depending on the applicationand are selected in accordance with the general binding methods knownincluding those referred to in: Maniatis et al. Molecular Cloning: ALaboratory Manual (2nd Ed. Cold Spring Harbor, N.Y., 1989); Berger andKimmel Methods in Enzymology, Vol. 152, Guide to Molecular CloningTechniques (Academic Press, Inc., San Diego, Calif., 1987); Young andDavism, P.N.A.S, 80: 1194 (1983). Methods and apparatus for carrying outrepeated and controlled hybridization reactions have been described inU.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623each of which are incorporated herein by reference

Certain embodiments may employ nucleic acid amplification methods fordetecting HRS polynucleotide sequences. The term “amplification” or“nucleic acid amplification” refers to the production of multiple copiesof a target nucleic acid that contains at least a portion of theintended specific target nucleic acid sequence. The multiple copies maybe referred to as amplicons or amplification products. In certainembodiments, the amplified target contains less than the complete targetgene sequence (introns and exons) or an expressed target gene sequence(spliced transcript of exons and flanking untranslated sequences). Forexample, specific amplicons may be produced by amplifying a portion ofthe target polynucleotide by using amplification primers that hybridizeto, and initiate polymerization from, internal positions of the targetpolynucleotide. Preferably, the amplified portion contains a detectabletarget sequence that may be detected using any of a variety ofwell-known methods.

“Selective amplification” or “specific amplification,” as used herein,refers to the amplification of a target nucleic acid sequence accordingto the present invention wherein detectable amplification of the targetsequence is substantially limited to amplification of target sequencecontributed by a nucleic acid sample of interest that is being testedand is not contributed by target nucleic acid sequence contributed bysome other sample source, e.g., contamination present in reagents usedduring amplification reactions or in the environment in whichamplification reactions are performed.

By “amplification conditions” is meant conditions permitting nucleicacid amplification according to the present invention. Amplificationconditions may, in some embodiments, be less stringent than “stringenthybridization conditions” as described herein. Oligonucleotides used inthe amplification reactions of the present invention hybridize to theirintended targets under amplification conditions, but may or may nothybridize under stringent hybridization conditions. On the other hand,detection probes of the present invention typically hybridize understringent hybridization conditions. Acceptable conditions to carry outnucleic acid amplifications according to the present invention can beeasily ascertained by someone having ordinary skill in the art dependingon the particular method of amplification employed.

Many well-known methods of nucleic acid amplification requirethermocycling to alternately denature double-stranded nucleic acids andhybridize primers; however, other well-known methods of nucleic acidamplification are isothermal. The polymerase chain reaction (U.S. Pat.Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188), commonly referred toas PCR, uses multiple cycles of denaturation, annealing of primer pairsto opposite strands, and primer extension to exponentially increase copynumbers of the target sequence. In a variation called RT-PCR, reversetranscriptase (RT) is used to make a complementary DNA (cDNA) from mRNA,and the cDNA is then amplified by PCR to produce multiple copies of DNA.

As noted above, the term “PCR” refers to multiple amplification cyclesthat selectively amplify a target nucleic acid species. Included arequantitative PCR (qPCR), real-time PCR), reverse transcription PCR(RT-PCR) and quantitative reverse transcription PCR (qRT-PCR) is welldescribed in the art. The term “pPCR” refers to quantitative polymerasechain reaction, and the term “qRT-PCR” refers to quantitative reversetranscription polymerase chain reaction. qPCR and qRT-PCR may be used toamplify and simultaneously quantify a targeted cDNA molecule. It enablesboth detection and quantification of a specific sequence in a cDNA pool,such as a selected HRS gene or transcript.

The term “real-time PCR” may use DNA-binding dye to bind to alldouble-stranded (ds) DNA in PCR, causing fluorescence of the dye. Anincrease in DNA product during PCR therefore leads to an increase influorescence intensity and is measured at each cycle, thus allowing DNAconcentrations to be quantified. However, dsDNA dyes such as SYBR Greenwill bind to all dsDNA PCR products. Fluorescence is detected andmeasured in the real-time PCR thermocycler, and its geometric increasecorresponding to exponential increase of the product is used todetermine the threshold cycle (“Cr”) in each reaction.

The term “Ct Score” refers to the threshold cycle number, which is thecycle at which PCR amplification has surpassed a threshold level. Ifthere is a higher quantity of mRNA for a particular gene in a sample, itwill cross the threshold earlier than a lowly expressed gene since thereis more starting RNA to amplify. Therefore, a low Ct score indicateshigh gene expression in a sample and a high Ct score is indicative oflow gene expression.

Certain embodiments may employ the ligase chain reaction (Weiss, R.1991, Science 254: 1292), commonly referred to as LCR, which uses twosets of complementary DNA oligonucleotides that hybridize to adjacentregions of the target nucleic acid. The DNA oligonucleotides arecovalently linked by a DNA ligase in repeated cycles of thermaldenaturation, hybridization and ligation to produce a detectabledouble-stranded ligated oligonucleotide product.

In certain embodiments, other techniques may be used to evaluate RNAtranscripts of the transcripts from a particular cDNA library, includingmicroarray analysis (Han, M., et al., Nat Biotechnol, 19: 631-635, 2001;Bao, P., et al., Anal Chem, 74: 1792-1797, 2002; Schena et al., Proc.Natl. Acad. Sci. USA 93:10614-19, 1996; and Heller et al., Proc. Natl.Acad. Sci. USA 94:2150-55, 1997) and SAGE (serial analysis of geneexpression). Like MPSS, SAGE is digital and can generate a large numberof signature sequences. (see e.g., Velculescu, V. E., et al., TrendsGenet, 16: 423-425, 2000; Tuteja R. and Tuteja N. Bioessays. 2004August; 26(8):916-22), although orders of magnitude fewer than that areavailable from techniques such as MPSS.

In certain embodiments, the term “microarray” includes a “nucleic acidmicroarray” having a substrate-bound plurality of nucleic acids,hybridization to each of the plurality of bound nucleic acids beingseparately detectable. The substrate can be solid or porous, planar ornon-planar, unitary or distributed. Nucleic acid microarrays include allthe devices so called in Schena (ed.), DNA Microarrays: A PracticalApproach (Practical Approach Series), Oxford University Press (1999);Nature Genet. 21(1) (suppl.): 1-60 (1999); Schena (ed.), MicroarrayBiochip: Tools and Technology, Eaton Publishing Company/BioTechniquesBooks Division (2000). Nucleic acid microarrays may include asubstrate-bound plurality of nucleic acids in which the plurality ofnucleic acids are disposed on a plurality of beads, rather than on aunitary planar substrate, as described, for example, in Brenner et al.,Proc. Natl. Acad. Sci. USA 97(4): 1665-1670 (2000). Examples of nucleicacid microarrays may be found in U.S. Pat. Nos. 6,391,623, 6,383,754,6,383,749, 6,380,377, 6,379,897, 6,376,191, 6,372,431, 6,351,7126,344,316, 6,316,193, 6,312,906, 6,309,828, 6,309,824, 6,306,643,6,300,063, 6,287,850, 6,284,497, 6,284,465, 6,280,954, 6,262,216,6,251,601, 6,245,518, 6,263,287, 6,251,601, 6,238,866, 6,228,575,6,214,587, 6,203,989, 6,171,797, 6,103,474, 6,083,726, 6,054,274,6,040,138, 6,083,726, 6,004,755, 6,001,309, 5,958,342, 5,952,180,5,936,731, 5,843,655, 5,814,454, 5,837,196, 5,436,327, 5,412,087, and5,405,783, the disclosures of which are incorporated by reference.

Additional examples include nucleic acid arrays that are commerciallyavailable from Affymetrix (Santa Clara, Calif.) under the brand nameGeneChip™. Further exemplary methods of manufacturing and using arraysare provided in, for example, U.S. Pat. Nos. 7,028,629; 7,011,949;7,011,945; 6,936,419; 6,927,032; 6,924,103; 6,921,642; and 6,818,394.

The present invention as related to arrays and microarrays alsocontemplates many uses for polymers attached to solid substrates. Theseuses include gene expression monitoring, profiling, library screening,genotyping and diagnostics. Gene expression monitoring and profilingmethods and methods useful for gene expression monitoring and profilingare shown in U.S. Pat. Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860,6,040,138, 6,177,248 and 6,309,822. Genotyping and uses therefore areshown in U.S. Ser. Nos. 10/442,021, 10/013,598 (U.S. Application No.2003/0036069), and U.S. Pat. Nos. 5,925,525, 6,268,141, 5,856,092,6,267,152, 6,300,063, 6,525,185, 6,632,611, 5,858,659, 6,284,460,6,361,947, 6,368,799, 6,673,579 and 6,333,179. Other methods of nucleicacid amplification, labeling and analysis that may be used incombination with the methods disclosed herein are embodied in U.S. Pat.Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061, and 6,197,506.

As will be apparent to persons skilled in the art, certain embodimentsmay employ oligonucleotides, such as primers or probes, foramplification or detection, as described herein. While the design andsequence of oligonucleotides depends on their function as describedherein, several variables are generally taken into account. Among themost relevant are: length, melting temperature (Tm), specificity,complementarity with other oligonucleotides in the system, G/C content,polypyrimidine (T, C) or polypurine (A, G) stretches, and the 3′-endsequence.

Certain embodiments therefore include methods for detecting a target HRSpolynucleotide in a sample, typically wherein the polynucleotidecomprises the sequence of a reference HRS polynucleotide describedherein, comprising a) hybridizing the sample with a probe comprising asequence complementary to the target polynucleotide in the sample, andwhich probe specifically hybridizes to said target polynucleotide, underconditions whereby a hybridization complex is formed between said probeand said target polynucleotide or fragments thereof, and b) detectingthe presence or absence of said hybridization complex, and optionally,if present, the amount thereof. Also included are methods for detectinga target HRS polynucleotide in a sample, the polynucleotide comprisingthe sequence of a reference HRS polynucleotide, as described herein,comprising a) amplifying the target polynucleotide or fragment thereof,and b) detecting the presence or absence of said amplified targetpolynucleotide or fragment thereof, and, optionally, if present, theamount thereof.

Embodiments of the present invention include a variety of HRSpolypeptide-based detection techniques, including antibody-baseddetection techniques. Included in these embodiments are the use of HRSpolypeptides to generate antibodies or other binders, which may then beused in diagnostic methods and compositions to detect or quantitateselected HRS polypeptides in a cell or other biological sample,typically from a subject.

Certain embodiments may employ standard methodologies such as westernblotting and immunoprecipitation, enzyme-linked immunosorbent assays(ELISA), flow cytometry, and immunofluorescence assays (IFA). Thesewell-known methods typically utilize one or more monoclonal orpolyclonal antibodies as described herein that specifically bind to aselected HRS polypeptide of the invention, or a unique region of thatHRS polypeptide, and generally do not bind significantly to other HRSpolypeptides, such as a full-length HRS polypeptide. In certainembodiments, the unique region of the HRS polypeptide may be encoded bya unique splice junction or a particular three-dimensional structure ofa newly identified alternate splice variant.

Certain embodiments may employ “arrays,” such as “microarrays.” Incertain embodiments, a “microarray” may also refer to a “peptidemicroarray” or “protein microarray” having a substrate-bound collectionor plurality of polypeptides, the binding to each of the plurality ofbound polypeptides being separately detectable. Alternatively, thepeptide microarray may have a plurality of binders, including but notlimited to monoclonal antibodies, polyclonal antibodies, phage displaybinders, yeast 2 hybrid binders, and aptamers, which can specificallydetect the binding of the HRS polypeptides described herein. The arraymay be based on autoantibody detection of these HRS polypeptides, asdescribed, for example, in Robinson et al., Nature Medicine 8(3):295-301(2002). Examples of peptide arrays may be found in WO 02/31463, WO02/25288, WO 01/94946, WO 01/88162, WO 01/68671, WO 01/57259, WO00/61806, WO 00/54046, WO 00/47774, WO 99/40434, WO 99/39210, and WO97/42507 and U.S. Pat. Nos. 6,268,210, 5,766,960, and 5,143,854, each ofwhich are incorporated by reference.

Certain embodiments may employ MS or other molecular weight-basedmethods for diagnostically detecting HRS polypeptide sequences. Massspectrometry (MS) refers generally to an analytical technique fordetermining the elemental composition of a sample or molecule. MS mayalso be used for determining the chemical structures of molecules, suchas peptides and other chemical compounds.

Generally, the MS principle consists of ionizing chemical compounds togenerate charged molecules or molecule fragments, and then measuringtheir mass-to-charge ratios. In an illustrative MS procedure: a sampleis loaded onto the MS instrument, and undergoes vaporization, thecomponents of the sample are ionized by one of a variety of methods(e.g., by impacting them with an electron beam), which results in theformation of positively charged particles, the positive ions are thenaccelerated by a magnetic field, computations are performed on themass-to-charge ratio (m/z) of the particles based on the details ofmotion of the ions as they transit through electromagnetic fields, and,detection of the ions, which in step prior were sorted according to m/z.

An illustrative MS instruments has three modules: an ion source, whichconverts gas phase sample molecules into ions (or, in the case ofelectrospray ionization, move ions that exist in solution into the gasphase); a mass analyzer, which sorts the ions by their masses byapplying electromagnetic fields; and a detector, which measures thevalue of an indicator quantity and thus provides data for calculatingthe abundances of each ion present.

The MS technique has both qualitative and quantitative uses, includingidentifying unknown compounds, determining the isotopic composition ofelements in a molecule, and determining the structure of a compound byobserving its fragmentation. Other uses include quantifying the amountof a compound in a sample or studying the fundamentals of gas phase ionchemistry (the chemistry of ions and neutrals in a vacuum). Accordingly,MS techniques may be used according to any of the methods providedherein to measure the presence or levels of an HRS polypeptide of theinvention in a biological sample, and to compare those levels to acontrol sample or a pre-determined value.

B. Discovery of Compounds and Therapeutic Agents

Certain embodiments relate to the use of HRS polypeptide or HRSpolynucleotide references sequences in drug discovery, typically toidentify agents that modulate one or more of the non-canonicalactivities of the reference HRS. For example, certain embodimentsinclude methods of identifying one or more “binding partners” of an HRSreference polypeptide, or a polypeptide that comprises an HRS referencesequence such as a cellular protein or other host molecule thatassociates with the HRS polypeptide and participates in itsnon-canonical activity or activities. Also included are methods ofidentifying a compound (e.g., polypeptide) or other agent that agonizesor antagonizes the non-canonical activity of an HRS referencepolypeptide or active variant thereof, such as by interacting with theHRS polypeptide and/or one or more of its cellular binding partners.

Certain embodiments therefore include methods of identifying a bindingpartner of an HRS reference polypeptide, comprising a) combining the HRSpolypeptide with a biological sample under suitable conditions, and b)detecting specific binding of the HRS polypeptide to a binding partner,thereby identifying a binding partner that specifically binds to the HRSreference polypeptide. Also included are methods of screening for acompound that specifically binds to an HRS reference polypeptide or abinding partner of the HRS polypeptide, comprising a) combining thepolypeptide or the binding partner with at least one test compound undersuitable conditions, and b) detecting binding of the polypeptide or thebinding partner to the test compound, thereby identifying a compoundthat specifically binds to the polypeptide or its binding partner. Incertain embodiments, the compound is a polypeptide or peptide. Incertain embodiments, the compound is a small molecule or other (e.g.,non-biological) chemical compound. In certain embodiments, the compoundis a peptide mimetic.

Any method suitable for detecting protein-protein interactions may beemployed for identifying cellular proteins that interact with an HRSreference polypeptide, interact with one or more of its cellular bindingpartners, or both. Examples of traditional methods that may be employedinclude co-immunoprecipitation, cross-linking, and co-purificationthrough gradients or chromatographic columns of cell lysates or proteinsobtained from cell lysates, mainly to identify proteins in the lysatethat interact with the HRS polypeptide.

In these and related embodiments, at least a portion of the amino acidsequence of a protein that interacts with an HRS polypeptide or itsbinding partner can be ascertained using techniques well known to thoseof skill in the art, such as via the Edman degradation technique. See,e.g., Creighton Proteins: Structures and Molecular Principles, W. H.Freeman & Co., N.Y., pp. 34 49, 1983. The amino acid sequence obtainedmay be used as a guide for the generation of oligonucleotide mixturesthat can be used to screen for gene sequences encoding such proteins.Screening may be accomplished, for example, by standard hybridization orPCR techniques, as described herein and known in the art. Techniques forthe generation of oligonucleotide mixtures and the screening are wellknown. See, e.g., Ausubel et al. Current Protocols in Molecular BiologyGreen Publishing Associates and Wiley Interscience, N.Y., 1989; andInnis et al., eds. PCR Protocols: A Guide to Methods and ApplicationsAcademic Press, Inc., New York, 1990.

Additionally, methods may be employed in the simultaneous identificationof genes that encode the binding partner or other polypeptide. Thesemethods include, for example, probing expression libraries, in a mannersimilar to the well known technique of antibody probing of lambda-gt11libraries, using labeled HRS protein, or another polypeptide, peptide orfusion protein, e.g., a variant HRS polypeptide or HRS domain fused to amarker (e.g., an enzyme, fluor, luminescent protein, or dye), or anIg-Fc domain.

One method that detects protein interactions in vivo is the two-hybridsystem. An example of this system has been described (Chien et al., PNASUSA 88:9578 9582, 1991) and is commercially available from Clontech(Palo Alto, Calif.). In certain instances, the two-hybrid system orother such methodology may be used to screen activation domain librariesfor proteins that interact with the “bait” gene product. By way ofexample, and not by way of limitation, an HRS reference polypeptide orvariant may be used as the bait gene product. An HRS binding partner mayalso be used as a “bait” gene product. Total genomic or cDNA sequencesare fused to the DNA encoding an activation domain. This library and aplasmid encoding a hybrid of a bait HRS gene product fused to theDNA-binding domain are co-transformed into a yeast reporter strain, andthe resulting transformants are screened for those that express thereporter gene.

Also included are three-hybrid systems, which allow the detection ofRNA-protein interactions in yeast. See, e.g., Hook et al., RNA.11:227-233, 2005. Accordingly, these and related methods can be used toidentify a cellular binding partner of an HRS polypeptide. These andrelated methods can also be used to identify other compounds such asbinding agents or nucleic acids that interact with the HRS polypeptide,its cellular binding partner, or both.

As noted above, once isolated, binding partners can be identified andcan, in turn, be used in conjunction with standard techniques toidentify proteins or other compounds with which it interacts. Certainembodiments thus relate to methods of screening for a compound thatspecifically binds to the binding partner of an HRS referencepolypeptide, comprising a) combining the binding partner with at leastone test compound under suitable conditions, and b) detecting binding ofthe binding partner to the test compound, thereby identifying a compoundthat specifically binds to the binding partner. In certain embodiments,the test compound is a polypeptide. In certain embodiments, the testcompound is a chemical compound, such as a small molecule compound orpeptide mimetic.

Certain embodiments include methods of screening for a compound thatmodulates the activity of an HRS reference polypeptide, comprising a)combining the polypeptide with at least one test compound underconditions permissive for the activity of the polypeptide, b) assessingthe activity of the polypeptide in the presence of the test compound,and c) comparing the activity of the polypeptide in the presence of thetest compound with the activity of the polypeptide in the absence of thetest compound, wherein a change in the activity of the polypeptide inthe presence of the test compound is indicative of a compound thatmodulates the activity of the polypeptide.

Certain embodiments include methods of screening for a compound thatmodulates the activity of a binding partner of an HRS referencepolypeptide, comprising a) combining the polypeptide with at least onetest compound under conditions permissive for the activity of thebinding partner, b) assessing the activity of the binding partner in thepresence of the test compound, and c) comparing the activity of thebinding partner in the presence of the test compound with the activityof the binding partner in the absence of the test compound, wherein achange in the activity of the binding partner in the presence of thetest compound is indicative of a compound that modulates the activity ofthe binding partner. Typically, these and related embodiments includeassessing a selected non-canonical activity that is associated with theHRS polypeptide or its binding partner. Included are in vitro and invivo conditions, such as cell culture conditions.

Certain embodiments include methods of screening a compound foreffectiveness as a full or partial agonist of an HRS referencepolypeptide or an active fragment or variant thereof, comprising a)exposing a sample comprising the polypeptide to a compound, and b)detecting agonist activity in the sample, typically by measuring anincrease in the non-canonical activity of the HRS polypeptide. Certainmethods include a) exposing a sample comprising a binding partner of theHRS polypeptide to a compound, and b) detecting agonist activity in thesample, typically by measuring an increase in the selected non-canonicalactivity of the HRS polypeptide. Certain embodiments includecompositions that comprise an agonist compound identified by the methodand a pharmaceutically acceptable carrier or excipient.

Also included are methods of screening a compound for effectiveness as afull or partial antagonist of an HRS reference polypeptide, comprisinga) exposing a sample comprising the polypeptide to a compound, and b)detecting antagonist activity in the sample, typically by measuring adecrease in the non-canonical activity of the HRS polypeptide. Certainmethods include a) exposing a sample comprising a binding partner of theHRS polypeptide to a compound, and b) detecting antagonist activity inthe sample, typically by measuring a decrease in the selectednon-canonical activity of the HRS polypeptide. Certain embodimentsinclude compositions that comprise an antagonist compound identified bythe method and a pharmaceutically acceptable carrier or excipient.

In certain embodiments, in vitro systems may be designed to identifycompounds capable of interacting with or modulating an HRS referencesequence or its binding partner. Certain of the compounds identified bysuch systems may be useful, for example, in modulating the activity ofthe pathway, and in elaborating components of the pathway itself. Theymay also be used in screens for identifying compounds that disruptinteractions between components of the pathway; or may disrupt suchinteractions directly. One exemplary approach involves preparing areaction mixture of the HRS polypeptide and a test compound underconditions and for a time sufficient to allow the two to interact andbind, thus forming a complex that can be removed from and/or detected inthe reaction mixture

In vitro screening assays can be conducted in a variety of ways. Forexample, an HRS polypeptide, a cellular binding partner, or testcompound(s) can be anchored onto a solid phase. In these and relatedembodiments, the resulting complexes may be captured and detected on thesolid phase at the end of the reaction. In one example of such a method,the HRS polypeptide and/or its binding partner are anchored onto a solidsurface, and the test compound(s), which are not anchored, may belabeled, either directly or indirectly, so that their capture by thecomponent on the solid surface can be detected. In other examples, thetest compound(s) are anchored to the solid surface, and the HRSpolypeptide and/or its binding partner, which are not anchored, arelabeled or in some way detectable. In certain embodiments, microtiterplates may conveniently be utilized as the solid phase. The anchoredcomponent (or test compound) may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized may be used toanchor the protein to the solid surface. The surfaces may be prepared inadvance and stored.

To conduct an exemplary assay, the non-immobilized component istypically added to the coated surface containing the anchored component.After the reaction is complete, un-reacted components are removed (e.g.,by washing) under conditions such that any specific complexes formedwill remain immobilized on the solid surface. The detection of complexesanchored on the solid surface can be accomplished in a number of ways.For instance, where the previously non-immobilized component ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the previously non-immobilizedcomponent is not pre-labeled, an indirect label can be used to detectcomplexes anchored on the surface; e.g., using a labeled antibodyspecific for the previously non-immobilized component (the antibody, inturn, may be directly labeled or indirectly labeled with a labeledanti-Ig antibody).

Alternatively, the presence or absence of binding of a test compound canbe determined, for example, using surface plasmon resonance (SPR) andthe change in the resonance angle as an index, wherein an HRSpolypeptide or a cellular binding partner is immobilized onto thesurface of a commercially available sensorchip (e.g., manufactured byBiacore™) according to a conventional method, the test compound iscontacted therewith, and the sensorchip is illuminated with a light of aparticular wavelength from a particular angle. The binding of a testcompound can also be measured by detecting the appearance of a peakcorresponding to the test compound by a method wherein an HRSpolypeptide or a cellular binding partner is immobilized onto thesurface of a protein chip adaptable to a mass spectrometer, a testcompound is contacted therewith, and an ionization method such asMALDI-MS, ESI-MS, FAB-MS and the like is combined with a massspectrometer (e.g., double-focusing mass spectrometer, quadrupole massspectrometer, time-of-flight mass spectrometer, Fourier transformationmass spectrometer, ion cyclotron mass spectrometer and the like).

In certain embodiments, cell-based assays, membrane vesicle-basedassays, or membrane fraction-based assays can be used to identifycompounds that modulate interactions in the non-canonical pathway of theselected HRS polypeptide. To this end, cell lines that express an HRSpolypeptide and/or a binding partner, or a fusion protein containing adomain or fragment of such proteins (or a combination thereof), or celllines (e.g., COS cells, CHO cells, HEK293 cells, Hela cells etc.) thathave been genetically engineered to express such protein(s) or fusionprotein(s) can be used. Test compound(s) that influence thenon-canonical activity can be identified by monitoring a change (e.g., astatistically significant change) in that activity as compared to acontrol or a predetermined amount.

For embodiments that relate to antisense and RNAi agents, for example,also included are methods of screening a compound for effectiveness inaltering expression of an HRS reference polynucleotide, comprising a)exposing a sample comprising the HRS reference polynucleotide to acompound such as a potential antisense oligonucleotide, and b) detectingaltered expression of the HRS polynucleotide. In certain non-limitingexamples, these and related embodiments can be employed in cell-basedassays or in cell-free translation assays, according to routinetechniques in the art. Also included are the antisense and RNAi agentsidentified by such methods.

Also included are any of the above methods, or other screening methodsknown in the art, which are adapted for high-throughput screening (HTS).HTS typically uses automation to run a screen of an assay against alibrary of candidate compounds, for instance, an assay that measures anincrease or a decrease in a non-canonical activity, as described herein.

C. Methods of Treatment

In another aspect, the present invention relates to methods of using thecompositions of the present invention for treating a cell, tissue orsubject with a composition as described herein. The cells or tissue thatmay be modulated by the present invention are preferably mammaliancells, or more preferably human cells. Such cells can be of a healthystate or of a diseased state.

Accordingly, the HRS agents described herein, including HRSpolypeptides, HRS polynucleotides, HRS polynucleotide-based vectors,antisense oligonucleotides, RNAi agents, as well as binding agents suchas peptides, antibodies and antigen-binding fragments, peptide mimeticsand other small molecules, can be used to treat a variety ofnon-limiting diseases or conditions associated with the non-canonicalactivities of a reference HRS. Examples of such non-canonical activitiesinclude modulation of cell proliferation, modulation of cell migration,modulation of cell differentiation (e.g., hematopoiesis), modulation ofapoptosis or other forms of cell death, modulation of cell signaling,modulation of angiogenesis, modulation of cell binding, modulation ofcellular metabolism, modulation of cytokine production or activity,modulation of cytokine receptor activity, modulation of inflammation,and the like.

Included are polynucleotide-based therapies, such as antisense therapiesand RNAi interference therapies, which typically relate to reducing theexpression of a target molecule, such as a particular splice variant ofan HRS polypeptide or a cellular binding partner of an HRS polypeptide,which otherwise contributes to its non-canonical activity. Antisense orRNAi therapies typically antagonize the non-canonical activity, such asby reducing expression of the HRS reference polypeptide. Also includedare polypeptides, antibodies, peptide mimetics, or other smallmolecule-based therapies, which either agonize or antagonize thenon-canonical activity of an HRS reference polypeptide, such as byinteracting directly with the HRS polypeptide, its cellular bindingpartner(s), or both.

In certain embodiments, for example, methods are provided for modulatingtherapeutically relevant cellular activities including, but not limitedto, cellular metabolism, cell differentiation, cell proliferation, celldeath, cell mobilization, cell migration, immune system function, genetranscription, mRNA translation, cell impedance, cytokine production,and the like, comprising contacting a cell with a HRS composition asdescribed herein. Accordingly, the HRS compositions may be employed intreating essentially any cell or tissue or subject that would benefitfrom modulation of one or more such activities.

The HRS compositions may also be used in any of a number of therapeuticcontexts including, for example, those relating to the treatment orprevention of neoplastic diseases, immune system diseases (e.g.,autoimmune diseases and inflammation), infectious diseases, metabolicdiseases, neuronal/neurological diseases, muscular/cardiovasculardiseases, diseases associated with aberrant hematopoiesis, diseasesassociated with aberrant angiogenesis, diseases associated with aberrantcell survival, and others.

For example, the compositions of the invention may be used asimmunomodulators for treating anti- or pro-inflammatory indications bymodulating the cells that mediate, either directly or indirectly,autoimmune and/or inflammatory disease, conditions and disorders. Theutility of the compositions of the invention as immunomodulators can bemonitored using any of a number of known and available techniques in theart including, for example, migration assays (e.g., using leukocytes,lymphocytes, monocytes), cytokine production assays, cell viabilityassays (e.g., using B-cells, T-cells, monocytes, NK cells), and thelike.

“Inflammation” refers generally to the biological response of tissues toharmful stimuli, such as pathogens, damaged cells (e.g., wounds), andirritants. The term “inflammatory response” refers to the specificmechanisms by which inflammation is achieved and regulated, including,merely by way of illustration, immune cell activation or migration,cytokine production, vasodilation, including kinin release,fibrinolysis, and coagulation, among others described herein and knownin the art. Ideally, inflammation is a protective attempt by the body toboth remove the injurious stimuli and initiate the healing process forthe affected tissue or tissues. In the absence of inflammation, woundsand infections would never heal, creating a situation in whichprogressive destruction of the tissue would threaten survival. On theother hand, excessive or chronic inflammation may associate with avariety of diseases, such as hay fever, atherosclerosis, and rheumatoidarthritis, among others described herein and known in the art.

Clinical signs of chronic inflammation are dependent upon duration ofthe illness, inflammatory lesions, cause and anatomical area affected.(see, e.g., Kumar et al., Robbins Basic Pathology-8^(th) Ed., 2009Elsevier, London; Miller, L M, Pathology Lecture Notes, AtlanticVeterinary College, Charlottetown, PEI, Canada). Chronic inflammation isassociated with a variety of pathological conditions or diseases,including, for example, allergies, Alzheimer's disease, anemia, aorticvalve stenosis, arthritis such as rheumatoid arthritis andosteoarthritis and arthritic gout, cancer, congestive heart failure,fibromyalgia, fibrosis, heart attack, kidney failure, lupus,pancreatitis, stroke, surgical complications, inflammatory lung disease,inflammatory bowel disease, atherosclerosis, neurological disorders,diabetes, metabolic disorders, obesity, and psoriasis, among othersdescribed herein and known in the art. Hence, HRS compositions may beused to treat or manage chronic inflammation, modulate any of one ormore of the individual chronic inflammatory responses, or treat any oneor more diseases or conditions associated with chronic inflammation.

Criteria for assessing the signs and symptoms of inflammatory and otherconditions, including for purposes of making differential diagnosis andalso for monitoring treatments such as determining whether atherapeutically effective dose has been administered in the course oftreatment, e.g., by determining improvement according to acceptedclinical criteria, will be apparent to those skilled in the art and areexemplified by the teachings of e.g., Berkow et al., eds., The MerckManual, 16^(th) edition, Merck and Co., Rahway, N.J., 1992; Goodman etal., eds., Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 10^(th) edition, Pergamon Press, Inc., Elmsford, N.Y.,(2001); Avery's Drug Treatment: Principles and Practice of ClinicalPharmacology and Therapeutics, 3rd edition, ADIS Press, Ltd., Williamsand Wilkins, Baltimore, Md. (1987); Ebadi, Pharmacology, Little, Brownand Co., Boston, (1985); Osolci al., eds., Remington's PharmaceuticalSciences, 18^(th) edition, Mack Publishing Co., Easton, Pa. (1990);Katzung, Basic and Clinical Pharmacology, Appleton and Lange, Norwalk,Conn. (1992).

Also included are methods of modulating an immune response, such as aninnate immune response. As used herein, the term “immune response”includes a measurable or observable reaction to an antigen, vaccinecomposition, or immunomodulatory molecule mediated by one or more cellsof the immune system. An immune response typically begins with anantigen or immunomodulatory molecule binding to an immune system cell. Areaction to an antigen or immunomodulatory molecule may be mediated bymany cell types, including a cell that initially binds to an antigen orimmunomodulatory molecule and cells that participate in mediating aninnate, humoral, cell-mediated immune response.

An “innate immune response,” as used herein, may involve binding ofpathogen-associated molecular patterns (PAMPs) or an HRS polypeptide tocell surface receptors, such as toll-like receptors. Activation oftoll-like receptors and Ipaf-signaling pathways in response to PAMPs orother signals leads to the production of immunomodulatory molecules,such as cytokines and co-stimulatory molecules, which induce and/orenhance an immune response. Cells involved in the innate immune responseinclude, for example, dendritic cells, macrophages, natural killercells, and neutrophils, among others.

Certain embodiments relate to increasing an innate immune response.Other embodiments relate to decreasing an innate immune response. Incertain aspects, an innate immune response is mediated by one or moretoll-like receptors (TLRs), such as TLR2 and/or TLR4. Certain FIRSpolypeptides of the invention bind to TLRS such as TLR2 and/or TLR4.TLRs recognize PAMPs that distinguish infectious agents from self andmediating the production of immunomodulatory molecules, such ascytokines, necessary for the development of effective adaptive immunity(Aderem, A and Ulevitch, R. J. Nature 406: 782-787 (2000) andBrightbill, H. D., Immunology 101: 1-10 (2000), herein incorporated byreference). Members of the toll-like receptor family recognize a varietyof antigen types and can discriminate between pathogens. For example,TLR2 recognizes various fungal, Gram-positive, and mycobacterialcomponents, TLR4 recognizes the Gram-negative product lipopolysaccharide(LPS), and TLR9 recognizes nucleic acids such as CpG repeats inbacterial DNA.

HRS compositions that stimulate innate immunity (e.g., via TLR2 and/rTLR4) can be useful in the treatment of a wide variety of conditions,either alone or in combination with other therapies. Specific examplesof such conditions include infectious diseases, such as bacterial,viral, and parasitic infectious diseases. HRS compositions thatstimulate innate immunity can also be useful as vaccine adjuvants, toenhance a subject's immune response to the primary antigen, whether in alive, attenuated, or other type of vaccine.

Examples of viral infectious diseases or agents (and their correspondingvaccines) include, but are not limited to, Hepatitis A, Hepatitis B,Hepatitis C, Hepatitis E, Caliciviruses associated diarrhoea, Rotavirusdiarrhoea, Haemophilus influenzae B pneumonia and invasive disease,influenza, measles, mumps, rubella, Parainfluenza associated pneumonia,Respiratory syncytial virus (RSV) pneumonia, Severe Acute RespiratorySyndrome (SARS), Human papillomavirus, Herpes simplex type 2 genitalulcers, HIV/AIDS, Dengue Fever, Japanese encephalitis, Tick-borneencephalitis, West-Nile virus associated disease, Yellow Fever,Epstein-Barr virus, Lassa fever, Crimean-Congo haemorrhagic fever, Ebolahaemorrhagic fever, Marburg haemorrhagic fever, Rabies, Rift Valleyfever, Smallpox, leprosy, upper and lower respiratory infections,poliomyelitis, among others described elsewhere herein.

Examples of bacterial infections disease or agents include, but are notlimited to, Bacillus antracis, Borellia burgdorferi, Brucella abortus,Brucella canus, Brucella melitensis, Brucella suis, Campylobacterjejuni, Chlamydia pneumoniae, Chlamydia psitacci, Chlamydia trachomatis,Clostridium botulinum, C. difficile, C. perfringens, C. tetani,Corynebacterium diphtheriae (i.e., diphtheria), Enterococcus,Escherichia coli, Haemophilus influenza, Helicobacter pylori, Legionellapneumophila, Leptospira, Listeria monocytogenes, Mycobacterium leprae,M. tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhea, N.meningitidis, Pseudomonas aeruginosa, Rickettsia recketisii, Salmonellatyphi, S. typhimurium, Shigella sonnei, Staphylococcus aureus, S.epidermidis, S. saprophyticus, Streptococcus agalactiae, S. pneumoniae,S. pyogenes, Treponema pallidum, Vibrio cholera, Yersinia pestis,Bordatella pertussis, and otitis media (e.g., often caused byStreptococcus pneumoniae, Haemophilus influenzae, or Moraxellacatarrhalis), among others described elsewhere herein.

Examples of parasitic infectious diseases include, but are not limitedto, Amoebiasis (e.g., Entemoeba histolytica), Hookworm Disease (e.g.,nematode parasites such as Necator americanus and Ancylostomaduodenale), Leishmaniasis, Malaria (four species of the protozoanparasite Plasmodium; P. falciparum, P. vivax, P. ovale, and P.malariae), Schistosomiasis (parasitic Schistosoma; S. mansoni, S.haematobium, and S. japonicum), Onchocerca volvulus (River blindness),Trypanosoma cruzi (Chagas disease/American sleeping sickness), andDracunculus medinensis, lymphatic filariasis.

Certain HRS compositions may be useful in the treatment or reduction ofendotoxic shock, which often results from exposure to foreign antigens,such as lipopolysaccharide (LPS). Because endotoxic shock can bemediated by TLR signaling, and naturally-occurring endogenous HRSfragments (e.g., SV9) may stimulate TLRs, certain of the binding agents,antisense agents, or RNAi agents provided herein may render a subjectmore resistant to endotoxic shock by antagonizing or otherwise reducingthe endogenous HRS fragment-mediated stimulation of TLR2 and/or TLR4.

Certain HRS compositions may be useful in reducing or antagonizingcertain immune activities. For instance, given the role of TLRs inmodulating cell migration, such as monocyte migration, HRS compositionsthat signal through TLRs may also modulate cell migration. In certainaspects, HRS compositions reduce or antagonize CCL1 mediated activities,such as immune cell migration, including monocyte migration. As oneexample, certain HRS compositions may activate TLRs, such as TLR2 and/orTLR4, which in certain instances leads to cytokine secretion (e.g.,MIP1α), and down-regulation in the levels or activity of relatedcytokine receptors (e.g., CCL1). Hence, HRS compositions may be employedto modulate immune activity such as cell migration associated with TLRsand cytokine receptors such as CCL1, and thereby treat TLR and/orCCR1-mediated diseases or conditions.

Also included are methods of treating immune diseases. Illustrativeimmune system diseases, disorders or conditions that may be treatedaccording to the present invention include, but are not limited to,primary immunodeficiencies, immune-mediated thrombocytopenia, Kawasakisyndrome, bone marrow transplant (for example, recent bone marrowtransplant in adults or children), chronic B cell lymphocytic leukemia,HIV infection (for example, adult or pediatric HIV infection), chronicinflammatory demyelinating polyneuropathy, post-transfusion purpura, andthe like.

Additionally, further diseases, disorders and conditions includeGuillain-Barre syndrome, anemia (for example, anemia associated withparvovirus B19, patients with stable multiple myeloma who are at highrisk for infection (for example, recurrent infection), autoimmunehemolytic anemia (for example, warm-type autoimmune hemolytic anemia),thrombocytopenia (for example, neonatal thrombocytopenia), andimmune-mediated neutropenia), transplantation (for example,cytomegalovirus (CMV)-negative recipients of CMV-positive organs),hypogammaglobulinemia (for example, hypogammaglobulinemic neonates withrisk factor for infection or morbidity), epilepsy (for example,intractable epilepsy), systemic vasculitic syndromes, myasthenia gravis(for example, decompensation in myasthenia gravis), dermatomyositis, andpolymyositis.

Further autoimmune diseases, disorders and conditions include but arenot limited to, autoimmune hemolytic anemia, autoimmune neonatalthrombocytopenia, idiopathic thrombocytopenia purpura,autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,dermatitis, allergic encephalomyelitis, myocarditis, relapsingpolychondritis, rheumatic heart disease, glomerulonephritis (forexample, IgA nephropathy), multiple sclerosis, neuritis, uveitisophthalmia, polyendocrinopathies, purpura (for example,Henloch-Scoenlein purpura), Reiter's disease, stiff-man syndrome,autoimmune pulmonary inflammation, Guillain-Barre Syndrome, insulindependent diabetes mellitus, and autoimmune inflammatory eye disease.

Additional autoimmune diseases, disorders or conditions include, but arenot limited to, autoimmune thyroiditis; hypothyroidism, includingHashimoto's thyroiditis and thyroiditis characterized, for example, bycell-mediated and humoral thyroid cytotoxicity; SLE (which is oftencharacterized, for example, by circulating and locally generated immunecomplexes); Goodpasture's syndrome (which is often characterized, forexample, by anti-basement membrane antibodies); pemphigus (which isoften characterized, for example, by epidermal acantholytic antibodies);receptor autoimmunities such as, for example, Graves' disease (which isoften characterized, for example, by antibodies to a thyroid stimulatinghormone receptor; myasthenia gravis, which is often characterized, forexample, by acetylcholine receptor antibodies); insulin resistance(which is often characterized, for example, by insulin receptorantibodies); autoimmune hemolytic anemia (which is often characterized,for example, by phagocytosis of antibody-sensitized red blood cells);and autoimmune thrombocytopenic purpura (which is often characterized,for example, by phagocytosis of antibody-sensitized platelets).

Further autoimmune diseases, disorders or conditions include, but arenot limited to, rheumatoid arthritis (which is often characterized, forexample, by immune complexes in joints); scleroderma with anti-collagenantibodies (which is often characterized, for example, by nucleolar andother nuclear antibodies); mixed connective tissue disease, (which isoften characterized, for example, by antibodies to extractable nuclearantigens, for example, ribonucleoprotein); polymyositis/dermatomyositis(which is often characterized, for example, by nonhistone anti-nuclearantibodies); pernicious anemia (which is often characterized, forexample, by antiparietal cell, antimicrosome, and anti-intrinsic factorantibodies); idiopathic Addison's disease (which is often characterized,for example, by humoral and cell-mediated adrenal cytotoxicity);infertility (which is often characterized, for example, byantispennatozoal antibodies); glomerulonephritis (which is oftencharacterized, for example, by glomerular basement membrane antibodiesor immune complexes); by primary glomerulonephritis, by IgA nephropathy;bullous pemphigoid (which is often characterized, for example, by IgGand complement in the basement membrane); Sjogren's syndrome (which isoften characterized, for example, by multiple tissue antibodies and/orthe specific nonhistone antinuclear antibody (SS-B)); diabetes mellitus(which is often characterized, for example, by cell-mediated and humoralislet cell antibodies); and adrenergic drug resistance, includingadrenergic drug resistance with asthma or cystic fibrosis (which isoften characterized, for example, by beta-adrenergic receptorantibodies).

Still further autoimmune diseases, disorders or conditions include, butare not limited to chronic active hepatitis (which is oftencharacterized, for example by smooth muscle antibodies); primary biliarycirrhosis (which is often characterized, for example, byanti-mitochondrial antibodies); other endocrine gland failure (which ischaracterized, for example, by specific tissue antibodies in somecases); vitiligo (which is often characterized, for example, byanti-melanocyte antibodies); vasculitis (which is often characterized,for example, by immunoglobulin and complement in vessel walls and/or lowserum complement); post-myocardial infarction conditions (which areoften characterized, for example, by anti-myocardial antibodies);cardiotomy syndrome (which is often characterized, for example, byanti-myocardial antibodies); urticaria (which is often characterized,for example, by IgG and IgM antibodies to IgE); atopic dermatitis (whichis often characterized, for example, by IgG and IgM antibodies to IgE);asthma (which is often characterized, for example, by IgG and IgMantibodies to IgE); inflammatory myopathies; and other inflammatory,granulomatous, degenerative, and atrophic disorders.

Also included are methods of modulating hematopoiesis and relatedconditions. Examples of hematopoietic processes that may be modulated bythe HRS polypeptides of the invention include, without limitation, theformation of myeloid cells (e.g., erythroid cells, mast cellsmonocytes/macrophages, myeloid dendritic cells, granulocytes such asbasophils, neutrophils, and eosinophils, megakaryocytes, platelets) andlymphoid cells (e.g., natural killer cells, lymphoid dendritic cells,B-cells, and T-cells). Certain specific hematopoietic processes includeerythropoiesis, granulopoiesis, lymphopoiesis, megakaryopoiesis,thrombopoiesis, and others. Also included are methods of modulating thetrafficking or mobilization of hematopoietic cells, includinghematopoietic stem cells, progenitor cells, erythrocytes, granulocytes,lymphocytes, megakaryocytes, and thrombocytes.

The methods of modulating hematopoiesis may be practiced in vivo, invitro, ex vivo, or in any combination thereof. These methods can bepracticed on any biological sample, cell culture, or tissue thatcontains hematopoietic stem cells, hematopoietic progenitor cells, orother stem or progenitor cells that are capable of differentiating alongthe hematopoietic lineage (e.g., adipose tissue derived stem cells). Forin vitro and ex vivo methods, stem cells and progenitor cells, whetherof hematopoietic origin or otherwise, can be isolated and/or identifiedaccording to the techniques and characteristics described herein andknown in the art.

In still other embodiments, the HRS compositions of the invention may beused to modulate angiogenesis, e.g., via modulation of endothelial cellproliferation and/or signaling. Endothelial cell proliferation and/orcell signaling may be monitored using an appropriate cell line (e.g.,Human microvascular endothelial lung cells (HMVEC-L) and Human umbilicalvein endothelial cells (HUVEC)), and using an appropriate assay (e.g.,endothelial cell migration assays, endothelial cell proliferationassays, tube-forming assays, matrigel plug assays, etc.), many of whichare known and available in the art.

Therefore, in related embodiments, the compositions of the invention maybe employed in the treatment of essentially any cell or tissue orsubject that would benefit from modulation of angiogenesis. For example,in some embodiments, a cell or tissue or subject experiencing orsusceptible to angiogenesis (e.g., an angiogenic condition) may becontacted with a suitable composition of the invention to inhibit anangiogenic condition. In other embodiments, a cell or tissueexperiencing or susceptible to insufficient angiogenesis (e.g., anangiostatic condition) may be contacted with an appropriate compositionof the invention in order to interfere with angiostatic activity and/orpromote angiogenesis.

Illustrative examples of angiogenic conditions include, but are notlimited to, age-related macular degeneration (AMD), cancer (both solidand hematologic), developmental abnormalities (organogenesis), diabeticblindness, endometriosis, ocular neovascularization, psoriasis,rheumatoid arthritis (RA), and skin disclolorations (e.g., hemangioma,nevus flammeus or nevus simplex). Examples of anti-angiogenic conditionsinclude, but are not limited to, cardiovascular disease, restenosis,tissue damage after reperfusion of ischemic tissue or cardiac failure,chronic inflammation and wound healing.

In other embodiments, the HRS compositions of the invention may be usedto modulate cellular proliferation and/or survival and, accordingly, fortreating or preventing diseases, disorders or conditions characterizedby abnormalities in cellular proliferation and/or survival. For example,in certain embodiments, the HRS compositions may be used to modulateapoptosis and/or to treat diseases or conditions associated withabnormal apoptosis. Apoptosis is the term used to describe the cellsignaling cascade known as programmed cell death. Various therapeuticindications exist for molecules that induce apoptosis (e.g. cancer), aswell as those that inhibit apoptosis (i.e. stroke, myocardialinfarction, sepsis, etc.). Apoptosis can be monitored by any of a numberof available techniques known and available in the art including, forexample, assays that measure fragmentation of DNA, alterations inmembrane asymmetry, activation of apoptotic caspases and/or release ofcytochrome C and AIF.

Illustrative diseases associated with increased cell survival, or theinhibition of apoptosis include, but are not limited to, cancers (suchas follicular lymphomas, carcinomas, and hormone-dependent tumors,including, but not limited to colon cancer, cardiac tumors, pancreaticcancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinalcancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Graves' disease, Hashimoto's thyroiditis,autoimmune diabetes, biliary cirrhosis, Behcet's disease, Crohn'sdisease, polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis, autoimmune gastritis, autoimmune thrombocytopenicpurpura, and rheumatoid arthritis) and viral infections (such as herpesviruses, pox viruses and adenoviruses), inflammation, graft vs. hostdisease (acute and/or chronic), acute graft rejection, and chronic graftrejection.

Further illustrative diseases or conditions associated with increasedcell survival include, but are not limited to, progression and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (for example, acute lymphocytic leukemia,acute myelocytic leukemia, including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(for example, chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia), myelodysplastic syndrome polycythemia vera,lymphomas (for example, Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

Illustrative diseases associated with increased apoptosis include, butare not limited to, AIDS (such as HIV-induced nephropathy and HIVencephalitis), neurodegenerative disorders (such as Alzheimer's disease,Parkinson's disease, amyotrophic lateral sclerosis, retinitispigmentosa, cerebellar degeneration and brain tumor or prior associateddisease), autoimmune disorders such as multiple sclerosis, Sjogren'ssyndrome, Graves' disease, Hashimoto's thyroiditis, autoimmune diabetes,biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis,systemic lupus erythematosus, immune-related glomerulonephritis,autoimmune gastritis, thrombocytopenic purpura, and rheumatoidarthritis, myelodysplastic syndromes (such as aplastic anemia), graftvs. host disease (acute and/or chronic), ischemic injury (such as thatcaused by myocardial infarction, stroke and reperfusion injury), liverinjury or disease (for example, hepatitis related liver injury,cirrhosis, ischemia/reperfusion injury, cholestosis (bile duct injury)and liver cancer), toxin-induced liver disease (such as that caused byalcohol), septic shock, ulcerative colitis, cachexia, and anorexia.

In still further embodiments, the compositions of the invention may beused in the treatment of neuronal/neurological diseases or disorders,illustrative examples of which include Parkinson's disease, Alzheimer'sdisease, Pick's disease, Creutzfeldt-Jacob disease, Huntington's chorea,alternating hemiplegia, amyotrophic lateral sclerosis, ataxia, cerebralpalsy, chronic fatigue syndrome, chronic pain syndromes, congenitalneurological anomalies, cranial nerve diseases, delirium, dementia,demyelinating diseases, dysautonomia, epilepsy, headaches, Huntington'sdisease, hydrocephalus, meningitis, movement disorders, muscle diseases,nervous system neoplasms, neurocutaneous syndromes, neurodegenerativediseases, neurotoxicity syndromes, ocular motility disorders, peripheralnervous system disorders, pituitary disorders, porencephaly, Rettsyndrome, sleep disorders, spinal cord disorders, stroke, sydenham'schorea, tourette syndrome, nervous system trauma and injuries, etc. Incertain embodiments, the neurological condition is associated with6-hydroxydopamine (6-OHDA)-induced neuron death, a neurotoxin that isthought to be involved in pathogenesis of certain neurologicalconditions such as Parkinson's disease, or a related mechanism.

Furthermore, additional embodiments relate to the use of thecompositions of the invention in the treatment of metabolic disorderssuch as adrenoleukodystrophy, Krabbe's disease (globoid cellleukodystrophy), metachromatic leukodystrophy, Alexander's disease,Canavan's disease (spongiform leukodystrophy), Pelizaeus-Merzbacherdisease, Cockayne's syndrome, Hurler's disease, Lowe's syndrome, Leigh'sdisease, Wilson's disease, Hallervorden-Spatz disease, Tay-Sachsdisease, etc. The utility of the compositions of the invention inmodulating metabolic processes may be monitored using any of a varietyof techniques known and available in the art including, for example,assays which measure adipocyte lipogenesis or adipocyte lipolysis.

In more specific embodiments of the invention, the HRS compositions ofthe invention may be used to modulate cellular signaling, for example,via cell signaling proteins. Cell signaling may be monitored using anyof a number of well known assays. For example, the induction of generalcell signaling events can be monitored through altered phosphorylationpatterns of a variety of target proteins. Detection of cell signalingactivities in response to treatment of cells with HRS polypeptidestherefore serves as an indicator of distinct biological effects. Targetproteins used for this assay may be selected so as to encompass keycomponents of major cellular signaling cascades, thereby providing abroad picture of the cell signaling landscape and its therapeuticrelevance. Generally, such assays involve cell treatment with HRSpolypeptides followed by immunodetection with antibodies thatspecifically detect the phosphorylated (activated) forms of the targetproteins.

Illustrative target proteins useful for monitoring therapeuticallyrelevant cell signaling events may include, but are not limited to: p38MAPK (mitogen-activated protein kinase; activated by cellular stress andinflammatory cytokines; involved in cell differentiation and apoptosis);SAPK/JNK (stress-activated protein kinase/Jun-amino-terminal kinase;activated by cellular stresses and inflammatory cytokines); Erk1/2,p44/42 MAPK (mitogen-activated protein kinase Erk1 and Erk2; activatedby wide variety of extracellular signals; involved in regulation of cellgrowth and differentiation); and Akt (activated by insulin and variousgrowth or survival factors; involved in inhibition of apoptosis,regulation of glycogen synthesis, cell cycle regulation and cellgrowth). General phosphorylation of tyrosine residues may also bemonitored as a general indicator of changes in cell signaling mediatedby phosphorylation.

Of course, it will be recognized that other classes of proteins, such ascell adhesion molecules (e.g., cadherins, integrins, claudins, catenins,selectins, etc.) and/or ion channel proteins may also be assayed formonitoring cellular events or activities modulated by the compositionsof the invention.

In other specific embodiments of the invention, the HRS compositions ofthe invention may be used to modulate cytokine production by cells, forexample, by leukocytes. Cytokine production may be monitored using anyof a number of assays known in the art (i.e., RT-PCR, ELISA, ELISpot,flow cytometry, etc.). Generally, such assays involve cell treatmentwith HRS polypeptides followed by detection of cytokine mRNA orpolypeptides to measure changes in cytokine production. Detection ofincreases and/or decreases in cytokine production in response totreatment of cells with HRS polypeptides therefore serves as anindicator of distinct biological effects. HRS polypeptides of theinvention may induce, enhance, and/or inhibit an immune or inflammatoryresponse by modulating cytokine production. For example, HRSpolypeptides and compositions of the invention may be used to alter acytokine profile (i.e., type 1 vs. type 2) in a subject. Illustrativecytokines that may measured for monitoring biological effects of the HRScompositions include, but are not limited IL-1α, IL-1β, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18, IL-23 TGF-β, TNF-α,IFN-α, IFN-β, IFN-γ, RANTES, MIP-1α, MIP-1β, MCP-1, GM-CSF, G-CSF, etc.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. The following examples are provided byway of illustration only and not by way of limitation. Those of skill inthe art will readily recognize a variety of noncritical parameters thatcould be changed or modified to yield essentially similar results.

EXAMPLES Example 1 Identification of Alternative Splice Variant,HRS-SV9, of the Human Histidyl-TRNA Synthetase (HRS) Gene

An alternative splice variant of the HRS gene, referred to as HRS-SV9,was identified by PCR as follows. A pair of primers, one covering theboundary region of 5′-untranslated region and Exon 1 (HRS-BPF:AGTGGACAGCCGGGATGGCAGAGC (SEQ ID NO: 1)) and the other near the 3′-endof Exon 4 (HRS-P1R: CAGGAAGTCGCCTATCTGAAG (SEQ ID NO: 2)), were used tosearch for an Intron 2 retention splicing event previously reported bythe University of California Santa Cruz EST database (EST#BP267368). PCRreaction products using cDNA as template gave rise to one distinct band(FIG. 1B, upper arrow) larger than the reference band (FIG. 1B, lowerarrow), which is the fragment amplified from the full-length HRS gene(NM_002109.3; SEQ ID NO: 3). This larger band was detected in a humanskeletal muscle cDNA library, but not in cDNA libraries from HEK293T orIMR32 cells, indicating tissue specificity of this splice variant. Thisband was excised, and the DNA fragment was extracted with a gelpurification kit for sequencing.

DNA sequencing confirmed an insertion from Intron 2, as previouslyreported, however there was no evidence of the reported T>C mutation inExon 2, making this as a distinct alternative splice variant of HRS gene(FIG. 3A). The sequence inserted from Intron 2 introduces a stop codonimmediately after Exon 2, such that the encoded protein sequence hasonly the first 60 amino acids of the full length HRS protein (FIG. 3B).FIG. 3C shows the nucleic acid coding sequence (SEQ ID NO: 5) andencoded protein sequence (SEQ ID NO: 6) for the HRS-SV9 splice variant.

Example 2 Identification of Alternative Splice Variant, HRS-SV11, of theHuman Histidyl-TRNA Synthetase (HRS) Gene

In this example, another alternative splice variant of the HRS gene,referred to as HRS-SV11, was also identified. A pair of primers, onecovering the boundary of the 3′-end of 5′-UTR and 5′-end of Exon 1(HRS-BPF: AGTGGACAGCCGGGATGGCAGAGC (SEQ ID NO: 1)) and the othercovering residues in the 5′-end of 3′-UTR (HRS-3′-UTR:ATAGTGCCAGTCCCACTTCC (SEQ ID NO: 7)), was used. A distinct band (FIG.2B, lower arrow) smaller than the reference band (FIG. 2B, upper arrow)was observed following PCR amplification of cDNA. The band was excised,gel-purified and sequenced. DNA sequencing confirmed that this is asplice variant of the HRS gene containing a deletion from Exon 3 to Exon10 (FIG. 3A). This deletion causes no frame-shifting. Thus, at theprotein level, HRS-SV11 comprises the N-terminal WHEP domain and theC-terminal anticodon domain of the reference HRS protein, but theaminoacylation domain is missing (FIG. 3B).

FIG. 3D shows the nucleic acid coding sequence (SEQ ID NO: 8) andencoded protein sequence (SEQ ID NO: 9) for the HRS-SV11 splice variant.As shown in FIG. 2C, this transcript was found in total cDNA of humanadult brain, lung, skeletal muscle and THP-1, Jurkat cells.

Example 3 Splice Variants HRS-SV9 and HRS-SV11 May be NaturallyOccurring

To test the natural existence of these splice variants at the proteinlevel, total cell extract were immunoblotted with anti-HRS antibodies.Two commercial antibodies, a monoclonal antibody with an epitope fromamino acids 1-97 (Novus Biologicals) and a polyclonal antibody with anepitope in the 50-200 amino acids near the C-terminus (Abcam), weretested with a number of cell lines, including HEK293T, C2C12, and muscletissues from adult rats, including tibialis muscles (representing fastmuscle) and soleus muscles (representing slow muscle). Both the N- andC-terminal antibodies detected a band (lower arrows in FIGS. 4A and 4B)in HEK293T, adult rat tibialis and soleus muscle having a sizeconsistent with the predicted size of the HRS-SV11 splice variantpolypeptide.

Immunoprecipitation with the N-terminal antibody detected a band indifferentiated C2C12 myotubes (lower arrow in FIG. 5A), having a sizeconsistent with the predicted size of the HRS-SV9 splice variantpolypeptide. Bands were not detected in HEK293T cells or C2C12differentiated myoblasts.

Total cell lysate of HEK293T cells that over-express myc-tagged HRS-SV11was also used as a reference for endogenous HRS-SV11 protein, whichshould be slightly smaller than myc-tagged HRS-SV11. As shown in FIGS.5B and 5C, a band running around 20 kDa but slightly smaller thanmyc-tagged HRS-SV11 was detected in IMR32 cells by both antibodies(lower arrows in B and C), suggesting the presence endogenous HRS-SV11.

Example 4 Secretion of Splice Variants HRS-SV9 and HRS-SV11 from HEK293TCells when Overexpressed

In this example, HRS-SV9 and HRS-SV11 were forcefully expressed inHEK293T cells and tested to determine whether they were secreted fromthe cells.

For plasmid construction, the wild type HRS, HRS-SV9 and HRS-SV11 codingsequences were cloned into pCI-neo-myc-myc-C vector (Promega, Madison,Wis.) through EcoRI/XhoI, respectively. For secretion assays, HEK293Tcells were transfected when they reach 60-70% confluency. 1 μg DNA wasmixed with 125 μl Opti-MEM. 4 μl lipofectamine 2000 was mixed with 125μl Opti-MEM. After 5 minutes, DNA-Opti-MEM complex was added tolipofectamine 2000-Opti-MEM complex with gentle tapping several times.The mixture was incubated 20 minutes at room temperature and addeddrop-wise on top of cells. After gentle swirling, cells were returned tothe incubator. Culture medium was refreshed after 6-7 hours. 24 hoursafter transfection, medium was changed to Dulbecco's Modified EagleMedium (DMEM) without serum. Both medium and total cell extract wereharvested after another 6 hours of incubation. Proteins in media wereprecipitated with 20% TCA (trichloroacetic acid). Both media and totalcell extracts were resolved on NuPAGE Novex 4-12% Bis-Tris Gel(Invitrogen) and immunoblotted with the N-terminal HRS and tubulinantibodies (Invitrogen).

Using this approach, HRS-SV9 and HRS-SV11, as well as full-length HRS,were detected in media fractions, demonstrating they were secreted fromthe HEK293T cells (FIG. 6). In contrast, enhanced green fluorescentprotein (EGFP) was not secreted (see FIG. 6C). As shown in FIGS. 6A-B,tubulin was used as leaky control; tubulin is present in the total celllysate fraction, but is absent in medium fraction, demonstrating noleakiness in this experiment.

Example 5 Splice Variants HRS-SV9 and HRS-SV11 Increase IL-2 Secretionin Activated T-Cells

When antigen is presented by antigen presenting cells (APC), theearliest detectable response of T cell activation is the secretion ofcytokines, such as IL-2. Through autocrine secretion, IL-2 triggers Tcells proliferation, thereby generating cells required to eliminateantigen. Thus, regulators of IL-2 secretion serve as immunomodulatorsfor T lymphocyte-mediated immune responses.

Leukemia Jurkat T cells (ATCC No: TIB-152) are widely used for T cellactivation research, using IL-2 expression and release as an indicationof activation. For T cell activation, Jurkat T cells were stimulated byphorbol esters (PMA) and ionomycin (IOM). IL-2 secretion into media wasevaluated by ELISA. As expected, PMA and ionomycin stimulated Jurkat Tcells to release IL-2 in a dose dependent manner. As shown in FIG. 7,HRS-SV9 and HRS-SV11, when co-applied with PMA and IOM significantlyincreased IL-2 secretion. Thus, both HRS-SV9 and HRS-SV11 exhibitedunexpected immunomodulatory activity.

Example 6 Splice Variant HRS-SV9 Stimulates TNF-α Secretion in PBMCs

Peripheral blood mononuclear cells (PBMCs) were isolated from humanblood. The cells were resuspended in RPMI media with 10% FBS to 1×10⁶cells/mL. One million cells were treated for 24 hours with HRS-SV9 at6.25, 12.5, 25, 50, 100, and 250 nM. PBMCs were also treated withLipopolysaccharide (LPS) at 1 EU/mL, PBS, or 100 nM Negative ControlProtein 1 or 2. After 24 hours, cell supernatant was collected bycentrifugation at 2000×g for 10 min and evaluated in a TNF-α ELISA assay(R&D Systems; Cat. DTA00C).

As shown in FIG. 8, HRS-SV9 stimulated PBMCs to secrete TNF-α in a dosedependent manner. In contrast, cells treated with PBS or negativecontrol proteins secreted minimal or no TNF-α □□ (PBS, Neg. Ctrl. 1 andNeg. Ctrl. 2). LPS, a known inducer of TNF-α secretion, gave rise to apositive signal at 1 EU/ml. Although a minimal amount of LPS was presentin the HRS-SV9 protein (˜0.11 EU/mL at 250 nM), the TNF-α signalobserved for HRS-SV9 is above that which may be attributed to LPS. Theresults of this example demonstrate that HRS-SV9 acts as a modulator ofTNF-α secretion.

Example 7 Hrs-SV11 Protects Rat Cortical Neurons and PC12 Cells from6-OHDA-Induced Neuron Cell Death

Since HRS-SV11 transcript was identified from a neuronal cell line, thisprotein was tested in neuroprotection assays by using cultured primaryrat cortical neurons and PC12 cells. The assays used were: (1)6-hydroxydopamine (6-OHDA)-induced neuron death, a neurotoxin that isthought to be involved in pathogenesis of Parkinson's disease (PD); (2)Beta-amyloid (Aβ)-induced neuron death (using the Aβ₁₋₄₂ form), whichreproduces Alzheimer's disease; (3) L-glutamic acid-induced neurondeath, which is observed in many neurological diseases such as stroke;and (4) MPP+-induced PC12 cell death. These experimental models havebeen extensively studied and are considered to be physiologicallyrelevant to human neurological diseases.

Preparation of Recombinant Protein.

HRS-SV11 was cloned into pET20b vector (Novagen) at EcoRV/NotI siteswithout the stop codon. A polyhistidine tag (6×His) from the pET2obvector was added to the C-terminus of HRS-SV11 protein, allowingpurification with nickel-NTA beads. E. coli Rosetta strain (Novagen) wastransformed with the pET20b-HRS-SV11 plasmid and grew at 37° C. withvigorous shaking until OD₆₀₀ reached 0.6-0.8. 200 μM IPTG was added toinduce protein expression. Bacteria was grown at 16° C. overnight.

The bacteria were then pelleted, resuspended in 50 ml 1×Ni-NTA buffer(50 mM Tris, 300 mM NaCl, and 25 mM imidazole, pH8.0) with 1 tablet ofComplete EDTA-free protease inhibitor (Roche) and 300 mg lysozyme(Sigma), and placed on rotating wheel at 4° C. for 30 min. The lysatewas sonicated with 6×10″ pulses with 5″ brakes (increase amplitude from25% to 50% to 75% for 3 cycles). Then lysate was spin down at 14,000 rpmfor 45 min at 4° C. Supernatant was collected and incubate with 1 mlNi-NTA resin slurry for 10-15 min in a 50 ml column. After incubation,the protein-beads mixture was washed extensively with 1×Ni-NTA buffer(50 mM Tris, 300 mM NaCl, 25 mM Imidazole, pH8.0) supplemented with 0.1°A Triton X-114 to remove endotoxin from bacteria. Upon finishing wash,proteins were eluted in 10 ml (1×) NI-NTA elution buffer (50 mM Tris,300 mM NaCl, 300 mM Imidazole, pH8.0). Protein was dialyzed in 10 kDacutoff slide-a-lyzer (Pierce) against 2×PBS for three times, thenconcentrated with a 10 kDa cutoff Amicon centricon (Fisher).Concentrated protein was stored in 50% glycerol and 2 mM DTT at −20° C.HRS-SV14 and wild type HRS were cloned into pET21a vector and purifiedin the same way as HRS-SV11.

Cell Culture and Treatment.

PC12 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM)(Invitrogen) supplemented with 8% horse serum, 8% fetal bovine serum, 30IU/ml penicillin and 30 μg/ml streptomycin at 37° C., 5% CO₂. Forneuroprotection assays, PC12 cells were seeded into 24-well plates at1.25*10⁵ cells/well and used one day after seeding.

Primary rat cortical neurons were isolated from rat embryos at embryonicday 18 (E18) and cultured as described previously (Brewer et al., 1995)with some modifications. Briefly, the cortex was dissected out in Hank'sBalanced Salt Solution (HBSS) supplemented with 1 mM sodium pyruvate and10 mM HEPES (pH 7.4) without Ca²⁺ and Mg²⁺ (Gibco). After beingtrypsinized for 15 min at 37° C., treated cortex were washed withplating medium (DMEM with 10% horse serum, supplemented with 0.5 mMGlutaMAX, 100 U/ml penicillin and 100 μg/ml streptomycin), andtriturated for several times. The cells were allowed to settle down for3 min, and then pelleted by centrifugation at 1,200 rpm for 5 minutes.The cell pellet was suspended in 1 ml plating medium and counted.1.5×10⁵ cells were seeded in poly-L-lysine (0.1 mg/ml)-coated 24-wellculture plates and maintained at 37° C., 5% CO₂. After 4 hrs, platingmedium was replaced by culture medium (Neurobasal medium, supplementedwith 0.5 mM GlutaMAX and (1×) B27 supplement). Half medium was refreshedonce a week. Experiments were performed at 9 day in vitro (DIV 9) unlessmentioned otherwise. One day before experiments, half medium wasrefreshed.

In drug treatment studies, drugs were diluted in culture medium (but6-OHDA was diluted in H₂O), and then applied to cells. For monosodiumglutamate (MSG)-induced neurotoxicity, neurons were exposed to MSG for20 min, and washed once with culture medium and kept in fresh culturemedium. Memantine was added at the same time with MSG. Forbeta-amyloid-induced toxicity, Aβ₁₋₄₂ was incubated at 37° C. for a dayto form aggregates, then was added to cells. For hydrogenperoxide-induced toxicity, a tablet of H₂O₂ was dissolved in 12.5 ml H₂Oto make a stock, and then added to cells.

The EC₅₀ of each neurotoxin was determined and used in the followingexperiments. Neuron viability was measured by MTT and LDH assays. TheMTT assay is based on utilization of mitochondrial dehydrogenases ofviable cells, which cleave the tetrazolium ring of the MTT and yieldpurple MTT formazan crystals, while the LDH assay measures the releasedLDH in medium due to disruption of membrane integrity. For the MTTassay, MTT was added into the medium at a final concentration of 0.5mg/ml and incubated with cells for 2 hrs at 37° C., 5% CO₂. Themitochondrial dehydrogenase cleaves the MTT tetrazolium ring and yieldspurple formazan crystals. After incubation, medium was aspirated and 500μl dimethylsulphoxide (DMSO) was added to solubilize the formazancrystals. The microplate was gently shaken and incubated at 37° C. for 5min. Then, 100 μl solution was taken and filled in a 96-well microplate,and the absorbance was read at 570 nm (630 nm as reference) byspectrophotometer (BMG Labtech, Offenburg, Germany). The relativeabsorbance (OD570-OD630) was used as an indicator of cell viability.

For the LDH assay, released LDH in the medium was measured by LDH assaykit (Roche) following the product's instruction. Medium was collectedand spun down. The supernatant was saved and 100 μl was filled in a wellof a 96-well microplate (MP). 100 μl of freshly prepared reactionmixture was added to each well, and incubated with medium for 15 min atroom temperature. The microplate was kept from light after adding thereaction mixture. The absorbance of the samples at 492 nm was measuredafter 15 min by spectrophotometer (BMG Labtech, Offenburg, Germany). 200μl of the assay medium was used as a background control. 2% Triton X-100was used as a positive control since it permeabilizes all cell membraneand releases the maximal amount of LDH.

Among the three neuroprotection assays, it was found that HRS-SV11protected cortical neurons from 6-OHDA-induced neuron death in adose-dependent manner when these neurons were pre-treated with HRS-SV11for 24 hr (FIG. 9A), but had little to no effect on Aβ-, L-glutamicacid-, and MPP+-induced neuron death (FIG. 10).

HRS-SV11 was also tested in a 6-OHDA-induced PC12 cell death model,since this model is also relevant to PD and has been well characterized.Similar protection was observed against 6-OHDA after pre-treating PC12cells with HRS-SV11 for 24 hr (FIGS. 9B-C). In addition to 24 hrpre-treatment, HRS-SV11 also exerted neuroprotection with even 30 minpre-treatment (FIG. 5D), suggesting new protein synthesis may not benecessary for HRS-SV11's neuroprotection.

Example 8 HRS-SV11 Protect Neurons Through a Non-Extracellular Mechanism

The exact mechanism of neurotoxicity of 6-OHDA is not clearlyunderstood, but there's more evidence favor an extracellular mechanism,rather than an intracellular one (see Blum et al., 2000; Izumi et al.,2005; Hanrott et al., 2006). 6-OHDA, in the extracellular space, isauto-oxidized into p-quinone and reactive oxygen species, such ashydrogen peroxide (H₂O₂). To examine whether HRS-SV11, like otherantioxidants, protects PC12 cells through reducing H₂O₂ and p-quinone inthe extracellular space, a cell-free system was used to measureaccumulation of p-quinone in the presence or absence of HRS-SV11 bytaking advantage of a property of p-quinone that p-quinone has a uniqueabsorbance at 490 nm.

As shown in FIG. 11A, 6-OHDA was auto-oxidized in test tube with timeand gave an accumulation of p-quinone. Addition of HRS-SV11 did notreduce the amount of p-quinone generated, while vitamin C, a knownanti-oxidant, effectively blocked p-quinone's production. This resultindicates that HRS-SV11 does not prevent 6-OHDA's neurotoxicity bydirectly blocking generation of p-quinone in the extracellular space,but instead utilizes another mechanism.

It was also tested whether HRS-SV11 blocks H₂O₂-induced neuron death.H₂O₂ is another major oxidization product of 6-OHDA, and it has beenshown that 6-OHDA-induced PC12 cell death is blocked by catalase, mostlylikely by hydrolyzing H₂O₂ (see Hanrott et al., 2006). As shown in FIG.11B, H₂O₂ induced cortical neuron death dose-dependently, with an EC₅₀around 120 mM. However, pre-treating cortical neurons with HRS-SV11(from 1 nM up to 1 μM) did not promote neuronal survival as compared toH₂O₂ (120 mM) alone (FIG. 11C). This result suggests that HRS-SV11either did not prevent or reduce the generation of H₂O₂ in theextracellular space, or it did not interfere with the intracellulardeath pathway initiated by H₂O₂.

Most anti-oxidants are effective when co-applied with an oxidant. Forexample, glutathione abolishes p-quinone's toxicity and catalase blocksH₂O₂'s toxicity. To confirm previous results in view of this phenomenon,HRS-SV11 was applied simultaneously with 6-OHDA. As shown in FIG. 11D,co-application of HRS-SV11 with 6-OHDA showed no rescue effect in bothcortical neurons and PC12 cells.

A wash-out experiment was also performed; prior to the application of6-OHDA, neurons were washed with PBS and refreshed with culture mediumto remove any HRS-SV11 left after pre-incubation with HRS-SV11. Thisregime reduces the chance of any direct interference with 6-OHDA. Asshown in FIG. 11E, the washout did not affect HRS-SV11's protectivefunction. Taken together, these data strongly argued that HRS-SV11exerts neuronal protection through an alternate signaling mechanism,rather than by reducing 6-OHDA or its oxidized products extracellularly.

Example 9 HRS-SV11 Suppresses 6-OHDA-Induced Apoptosis of PC12 Cells

Apoptosis has been suggested as a main cause of 6-OHDA's toxicity (seeChoi et al., 1999; Blum et al., 2001). Nuclear fragmentation andcondensation is a hallmark of late apoptosis. Hoechst 33342 was used tostain and identify apoptotic cells after exposure to 6-OHDA in thepresence or absence of HRS-SV11. As shown in FIG. 12, around 20% PC12cells underwent apoptosis after 8 hrs exposure to 200 μM 6-OHDA, asdiscriminated by Hoechst staining. Pre-treatment with HRS-SV11 (at 1000nM) greatly reduced the portion of apoptotic cells (see FIG. 12D forapoptotic cell counts). This result suggests that HRS-SV11 protectedPC12 cells through suppression of apoptosis induced by 6-OHDA.

Example 10 Cysteine Residues Contribute to the Neuroprotective Effectsof HRS-SV11

HRS-SV11 contains three cysteines (Cys): Cys117, 169 and 171. Cys169 isthe last third amino acid and Cys171 is the last amino acid (see FIG.13A). Cys169 and Cys 171 were modified to achieve a homogenous monomerpopulation of HRS-SV11 for structural studies. Two mutants were made,(1) HRS-SV11_deIC and (2) HRS-SV11_C2S.

The HRS-SV11_deIC variant (referred to as delC) has the last three aminoacids (including Cys169 and Cys171) in HRS-SV11 deleted. TheHRS-SV11_C2S variant (referred to as C2S) has Cys169 and Cys171 mutatedto serine residues (Ser).

Analytical gel filtration chromatography was carried out on the proteinsusing an AKTA FPLC system (GE Healthcare). Protein samples were loadedonto a Superose 12 10/300 GL column (GE Healthcare) equilibrated with abuffer containing 50 mM Tris-HCl pH 7.5, 100 mM NaCl, and 1 mM DTT. Asanalyzed by analytical gel filtration (see FIG. 13B), HRS-SV11_C2S hadone peak, corresponding to the monomer form, and HRS-SV11_delC had twopeaks, a large one corresponding to monomer and a small onecorresponding to dimer. Cys 177 was also modified to Ser (the C117Svariant), and FIG. 13 shows that this mutation fixed HRS-SV11 in dimerform. Wild type HRS-SV11 was in a form between dimer and monomer,suggesting HRS-SV11 protein dynamically switched between dimer andmonomer.

The HRS-SV11 mutants were also tested in the 6-OHDA-induced PC12 celldeath model. As shown in FIG. 13C, the C2S and deIC mutants lost theprotective function observed with the wild type HRS-SV11 protein,indicating the importance of these two cysteines for HRS-SV11'sneuroprotection.

Example 11 HRS-SV11 Exerts Neuroprotection Through JAK2, JNK and P38

To explore the neuroprotective signaling pathway of HRS-SV11, a numberof specific chemical inhibitors were used to interfere with specificsignaling molecules. The results are shown in FIG. 14. From a panel ofinhibitors, it was found that inhibition of JAK2 (by AG490 at 40 μM),JNK and p38 together (by SB202190 at 10 μM and SP600125 at 10 μM),suppressed HRS-SV11's protective effect, while inhibiting phospholipaseC (PLC) by U73122, or MKK by arctigenin, or JNK or p38 alone, had noeffect in PC12 cells. These results suggest involvement of JAK2, JNK andp38 in HRS-SV11's signaling.

Example 12 HRS-SV11 Binds to CCR5

To further understand HRS-SV11's neuroprotection, potential cognatereceptors on cell surface were identified. An artificial fragmentcontaining amino acid 1-48 of HRS (1-48 a.a.), as well as wild type HRSprotein, induced CCR5-expressing HEK293T cells migration, but a deletionmutant without a.a. 1-48 did not (Howard et al., 2002). Since HRS-SV11contains the a.a.1-48 of wild-type HRS, CCR5 was tested as a candidatereceptor for HRS-SV11.

Human CCR5 was amplified and cloned into to pEGFP-N1 vector. Human CCR1receptor, also a CC chemokine receptor, was included as a control.HEK293T cells were transfected with CCR5-EGFP or CCR1-EGFP plasmids and1 d after transfection, cells were washed with PBS and detached fromculture dish by trypsin. 1×10^6 cells were put into FACS tube in 100 μlcomplete media and kept on ice (cells were kept cold for remainder ofthe assay). Cells were treated with recombinant HRS-SV11 protein for45′. Then cells were washed once with 1 ml staining buffer (1×PBS+3%FBS) and spin down at 4° C., 400×g for 10′. After wash, cells wereincubated with 0.3 μl primary anti-V5-FITC antibody (3 μg/ml) in 100 μlstaining buffer for 30′ in dark (kept in dark for remainder of theassay). Cells were then washed twice with 1 ml staining buffer, 4° C.,400×g for 10′ and spun down. After the final wash, cell pellets wereresuspended in 800 μl staining buffer and analyzed immediately by FACS.

Both CCR5-EGFP and CCR1-EGFP protein localized properly to cell membranewhen transfected into HEK293T cells as expected (data not shown). ByFluorescence-activated cell sorting (FACS), it was found thatapplication of recombinant HRS-SV11 protein to CCR5-expressing HEK293Tcells increased surface binding of His tag antibody as reflected by aright shift of the curve (FIG. 15B), but had no shift in CCR1-expressingcells (FIG. 15D), suggesting that CCR5 is a potential receptor forHRS-SV11. As shown in FIG. 15C, this shift was not affected bypre-treating CCR5-expressing HEK293T cells with Met-RANTES, a CCR5agonist. Since Met-RANTES binds to the N-terminus of CCR5 receptor, thisdata suggests that the N-terminus of CCR5 is not involved in HRS-SV11binding to CCR5.

Example 13 Identification of a Neuroprotective Alternative SpliceVariant, HRS-SV14, of the Human Histidyl-TRNA Synthetase (HRS) Gene

In this experiment, another splicing variant of HRS gene, namedHRS-SV14, was identified from human fetus brain by nested PCR (see FIG.16A, arrow). To set-up the first PCR reaction, a 10-ul reaction mixturewas generated containing 1 μl of first strand cDNA, 1× of Advantage 2PCR buffer (Clontech), 200 μM of each dNTP (Ambion), 250 pM of eachforwards and reverse primers (IDT oligo), and 1.25× of Advantage 2Polymerase Mix (Clontech). Primers for first PCR were hsH1-E2F1 (5′-TGAAAC TGA AGG CAC AGC TG-3′) (SEQ ID NO:12) and hsH1-E13R1 (5′-TCT TCT CTTCGG ACA TCC AC-3′) (SEQ ID NO:13). Thermo cycling conditions for firstPCR were 1 minute at 95° C. followed by 20 cycles of 20 seconds at 95°C., 30 seconds at 58° C. and 1 minute at 72° C., a final extension of 5minutes at 72° C. PCR set-up and thermo cycling conditions for nestedPCR were as the same as that for first PCR, except that, in nested PCR,template is 1000-fold diluted first PCR product, and primers arernH1-E02F1 (5′-AAC AGA AGT TCG TCC TCA AAA C-3′) (SEQ ID NO:14) andrnH1-E12J13R2(5′-TCC ACC TCT TCT CTG CTC GTC A-3′) (SEQ ID NO:15).Nested PCR products were resolved by electrophoresis. Distinct PCRproducts were isolated and purified by NucleoSpin Extract II kit(Macherey-Nagel). Isolated PCR products were cloned by using TOPO TACloning Kit for Sequencing (Invitrogen). Plasmids with PCR productssuccessfully inserted were obtained and sequenced. To identifyalternative splicing events, sequences of PCR products were aligned tohuman HARS mRNA sequence in NCBI database (Accession number: NM_002109).

Sequence alignment showed that HRS-SV14 transcript has a deletion of 894bp, from Exon 4 to Exon 10 of human HRS gene (FIG. 16B), which makesHRS-SV14 protein lack of amino acid 101-398 a.a. of the wild type HRSprotein, as shown in FIG. 16C. HRS-SV14 is similar to HRS-SV11, exceptthat it retains Exon 3, which translates into 40 a.a. of theaminoacylation domain.

Recombinant HRS-SV14 protein was then produced and tested forneuroprotective effects in the 6-OHDA-induced PC12 cell death model. Asshown in FIG. 16D, pre-treating PC12 cells with 500 nM HRS-SV14 for 24hrs significantly reduced PC12 cell death upon exposure to 6-OHDA, andthe level of neuroprotection was comparable to that of HRS-SV11.

Example 14 Histidyl-TRNA Synthetase Splice Variant SV9 Inhibits THP-1Migration

To characterize the properties of SV9, a splice variant of thehistidyl-tRNA synthetase (amino acids 1-60), a migration assay was setup based on a prior publication suggesting chemoattractant propertiesfor both the full length histidyl-tRNA synthetase and a fragment thereof(amino acids 1-48) [Howard et al. (2002), J. Exp. Med., 196:781-791].

THP-1 cells (ATCC, Catalog # TIB-202) were cultured in RPMI-1640 medium(ATCC, Catalog #30-2001) supplemented with 10% heat-inactivated FBS(Invitrogen, Catalog #10082147) and 0.05 mM 2-β-mercaptoethanol. Celldensity was kept at 2-4×10⁵ cells/ml. Before the migration assay, THP-1cells were collected by centrifugation, adjusted to a density of 6×10⁶cells/ml and starved for 45 minutes in migration buffer (RPMI-1640medium with 0.1% BSA) containing 6 μg/ml Calcein AM (Invitrogen, CatalogNo. C3099). At the same time, SV9 (or PBS as control) was added to thecells at different final concentrations. 100 μl cells (containing 6×10⁵cells) pre-treated with SV9 were added to the upper chamber of themigration apparatus. 600 μl migration buffer containing CCL-5 or PBSbuffer were added to the lower chamber and cells were allowed to migratefor 2 hours. Cells that migrated to the lower chamber were collected andresuspended in 100 μl PBS, transferred into 384-well opaque Greinerplate and fluorescence was read in a plate reader.

As shown in FIG. 19, SV9 unexpectedly inhibited migration of THP-1 cellsto other ligands such as CCL-5.

Example 15 Histidyl-TRNA Synthetase Splice Variant SV9 Inhibits CCR-1Mediated THP-1 Migration

To further characterize the properties of SV9, the migration assay wasused to determine which receptor SV9 engages to inhibit migration ofTHP-1 cells, since CCL-5 can potentially engage three receptors, CCR1,CCR3 and CCR5.

THP-1 cells (ATCC, Catalog # TIB-202) were cultured in RPMI-1640 medium(ATCC, Catalog #30-2001) supplemented with 10% heat-inactivated FBS(Invitrogen, Catalog #10082147) and 0.05 mM 2-β-mercaptoethanol. Celldensity was kept at 2-4×10⁵ cells/ml. Before the migration assay, THP-1cells were collected by centrifugation, adjusted to a density of 6×10⁶cells/ml and starved for 45 minutes in migration buffer (RPMI-1640medium with 0.1% BSA) containing 6 μg/ml Calcein AM (Invitrogen, CatalogNo. C3099). At the same time, SV9 (or PBS as control) was added to thecells at different final concentrations. 100 μl cells (containing 6×10⁵cells) pre-treated with SV9 were added to the upper chamber of themigration apparatus. 600 μl migration buffer containing CCL-23, a ligandwhose only know reactivity is toward CCR-1, or PBS buffer were added tothe lower chamber and cells were allowed to migrate for 2 hours. Cellsthat migrated to the lower chamber were collected and resuspended in 100μl PBS, transferred into 384-well opaque Greiner plate and fluorescencewas read in a plate reader.

FIG. 20 shows that SV9 inhibits migration of THP-1 cells to CCL-23, andtherefore likely inactivates the CCR1 receptor pathway.

Example 16 Histidyl-TRNA Synthetase Splice Variant SV9 ActivatesToll-Like Receptors

Previous findings [Parker et al. (2004), J. Immunol., 172:4977-4986]suggested that Toll-like receptor activation can downregulate CCR1. Weused a cell-based reporter assay to determine whether SV9 can alsoactivate Toll-like receptors.

RAW-Blue™ cells (InvivoGen, raw-sp) expressing various Toll-likereceptors were maintained in DMEM medium (Invitrogen) supplemented with10% FBS with 1×HEK-Blue™ Selection (InvivoGen, hb-sel). On the day ofthe assay, the medium was removed and the cells rinsed twice with PBS.The cells were trypsinized or scraped and resuspended in fresh growthmedium to prepare a cell suspension at approximately 550,000 cells/ml.20 μl of SV9, or controls, at various concentrations were added to thewells of a flat-bottom, 96-well plate including a negative control, suchas endotoxin-free water. 180 μl of cell suspension (approximately100,000 cells) were added per well and the plate was incubated at 37° C.in a 5% CO₂ incubator for 18-24 hours. The next day, a QUANTI-Blue™solution was prepared according to the manufacturer's instructions. 160μl of resuspended QUANTI-Blue™ were transferred to the wells of a96-well plate, followed by the addition of 40 μl of induced RAW-Blue™cell supernatant. The plate was then incubated at 37° C. for 30 minutesup to 6 hours. The levels of secreted alkaline phosphatase (SEAP) weredetermined using a spectrophotometer at 620-655 nm.

FIG. 21 shows that SV9 protein activates Toll-like receptors present atthe surface of the RAW-Blue™ cells, and stimulates production of theSEAP reporter.

Example 17 Histidyl-TRNA Synthetase Splice Variant SV9 PreferentiallyActivates Toll-Like Receptor 4

To further characterize which Toll-like receptor SV9 engages, cellsexpressing only TLR-2 or TLR-4 were used in a similar reporter assay.

TLR-2 or TLR-4 expressing 293 cells (InvivoGen, Catalog # hb2-cells)were maintained in DMEM medium (Invitrogen) supplemented with 10% FBSand 1×HEK-Blue™ Selection (InvivoGen, Catalog # hb-sel). On the day ofthe assay, 20 μl of SV9 at different concentrations, or controls, wereadded to the wells of a flat-bottom 96-well plate. A cell suspension ofHEK-Blue™-hTLR2 or TLR4 cells was prepared at 5×10⁵ cells per ml in TestMedium (DMEM, 10% heat-inactivated FBS). 90 μl of cell suspension(approximately 50,000 cells) were added per well and the plate wasincubated at 37° C. in a CO₂ incubator for 20-24 hours. The next day, aQUANTI-Blue™ solution was prepared according to the manufacturer'sinstructions. 180 μl of resuspended QUANTI-Blue™ (InvivoGen: Catalog #rep-qb1) were transferred to the wells of a 96-well plate, followed bythe addition of 20 μl of induced HEK-Blue™-hTLR2 or TLR4 cellsupernatant. The plate was then incubated at 37° C. for 1-3 hours. Thelevels of secreted alkaline phosphatase (SEAP) were determined using aspectrophotometer at 650 nm.

FIG. 22 shows that SV9 protein activates both TLR2 (22A) and TLR4 (22B)but is significantly more potent on TLR4.

Example 18 Histidyl-TRNA Synthetase Splice Variant SV9 StimulatesMIP-1-Alpha Secretion

Toll-like receptor activation has been shown to result in MIP-1αsecretion which, in turn, could trigger CCR1 down regulation [Parker etal. (2004), J. Immunol., 172:4977-4986]. We used an ELISA assay todetermine whether SV9 engagement of the TLR-2 and/or TLR-4 results inMIP-1α secretion.

THP-1 cells (ATCC, Catalog # TIB-202) were cultured in RPMI-1640 medium(ATCC, Catalog #30-2001) supplemented with 10% heat-inactivated FBS(Invitrogen, Catalog #10082147) and 0.05 mM 2-β-mercaptoethanol.

Cells were seeded at a density of 1×10⁶/ml in a 24 well plate and SV9 orLPS at different concentrations was added to each wells. After 24 hourincubation, the supernatant was collected from each well bycentrifugation at 250 g and removal of the supernatant. The expressionlevels of MIP-1α from each sample were determined by using a humanCCL3/MIP-1α Immunoassay Quantikine kit (R&D, Catalog # DMA00) accordingthe manufacturer's instructions.

FIG. 23 shows that SV9 stimulates the secretion of MIP-1α with kineticsdistinct from LPS.

The disclosure above is descriptive, illustrative and exemplary and isnot to be taken as limiting the scope defined by the appended claimswhich follow.

The invention claimed is:
 1. A method of reducing a cancer ormalignancy, comprising administering to a subject in need thereof acomposition comprising a pharmaceutically-acceptable carrier and anantibody or antigen-binding fragment thereof that exhibits bindingspecificity for a histidyl-tRNA synthetase (HRS) splice variantpolypeptide consisting of a sequence set forth in SEQ ID NO:6, 9, or 11,or a fragment of SEQ ID NO:6, 9, or 11, wherein the antibody orantigen-binding fragment thereof is at least about 90% pure.
 2. Themethod of claim 1, wherein the antibody or antigen-binding fragmentthereof antagonizes a non-canonical activity of the HRS splice variantpolypeptide.
 3. The method of claim 1, wherein the fragment of SEQ IDNO:6, 9, or 11 consists of the WHEP domain of HRS.
 4. The method ofclaim 1, wherein the fragment of SEQ ID NO:9 or 11 consists of theanticodon binding domain of HRS.
 5. The method of claim 1, wherein theantibody or antigen binding fragment thereof is a monoclonal antibody.6. The method of claim 1, wherein the antibody is a humanized antibody.7. The method of claim 1, wherein the antibody or antigen-bindingfragment thereof is an Fv fragment or a single chain Fv (sFv)polypeptide.
 8. The method of claim 1, wherein the composition is asterile injectable solution.
 9. The method of claim 1, wherein thecomposition is buffered and comprises an isotonic agent.
 10. The methodof claim 1, wherein the composition is suitable for intravenous,intramuscular, subcutaneous, or intraperitoneal administration.
 11. Themethod of claim 1, wherein the composition comprises a surfactant. 12.The method of claim 1, wherein the antibody or antigen-binding fragmentthereof is at least about 95% pure.